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Anal. Chem. 1990, 62, 1604-1610
Dual-Channel Flame Infrared Emission Detector for Gas Chromatography S. Ravishankar, D. C. Tilotta, S. W. Kubala, M.A. Busch, and K. W. Busch* Department of Chemistry, B. U. Box 7348, Baylor University, Waco, Texas 76798-7348
A dual-channel flame Infrared emlsslon (FIRE)detector for gas chromatography (QC) was developed to reduce the adverse effect of the additive ndse component of flame background on the signal-to-nolse ratio obtalned with the system. The dual-channel FIRE-GC detector employs a beam splitter to divide the radiation from the source Into two optical channels. The fadlation in each channel passes through different optlcal notch filters to select the bands associated with the appropriate background and the anatyte. The filtered beams are then monitored by separate lead selenlde detectors that form part of a Wheatstone bridge network whose output Is fed to the differentla1 (A-B) Input of a lock-in ampllfler. I t was determined that the fluctuations In the flame water background at 3.0 pm were well correlated wlth those In the flame water background at 4.4 pm and that the 3.0-pm emlssion would generate a dgnal that COUW be used to reduce both the absolute magnitude of the background as well as the background fiuctuatlons In the analytical channel. Three alternative methods of background subtraction are discussed, and experlmental results obtalned by uslng an optlcal attenuatlon method are presented. Use of the dual-channel arrangement was found to Improve the slgnal-to-nolse ratlo of the output by a factor of 3-5, compared with the singlechannel mode of operatlon. A linear dynamk range over 4.5 decades of concentratlon and a detection llmlt of 56 ng 8-’ were obtalned for Freon-113 (C&i,F,) by using the dualchannel FIRE detector in the subtracted mode. Comparison chromatograms udng the dual-channel FIRE detector and a thermal conductivity detector are presented.
INTRODUCTION Infrared emission from species present in hot gaseous sources such as optically thin combustion flames can form the basis for a promising laboratory method for the determination of infrared-active molecules (I). In their initial paper on the analytical applications of flame infrared emission (FIRE) spectroscopy, Hudson and Busch ( 2 ) showed how a hydrogen-air flame could be used for the dual purpose of oxidizing organic compounds to carbon dioxide and then vibrationally exciting the C 0 2produced. The vibrationally excited carbon dioxide molecules were detected by monitoring the intensity of the 0-C-0 asymmetric stretching vibration at 4.4 pm by using a lead selenide photoconductivity detector. Recent work ( 3 ) has shown that a number of classes of organic compounds produce combustion products in addition to carbon dioxide and water in the hydrogen-air flame. Many of these products also emit clearly defined, analytically useful infrared emission bands. In its most basic form, the FIRE detector consists of a hydrogen-air flame supported on a capillary burner, a CaFz lens to collect the infrared emission from the flame, a chopper to modulate the radiation, an optical notch filter to select the desired infrared emission band, and a lead selenide detector
with associated signal processing electronics. To date, the FIRE detector has been used as a detector in both gas and liquid chromatography ( 2 , 4 ) . Nonchromatographic applications of FIRE include the determination of total inorganic carbon (5) and volatile organics (1) in water samples. The determination of chloride ion and available chlorine in aqueous samples has also been demonstrated (6) by using the intensity of the infrared emission due to the stretching vibration of HCl. Recent work in this laboratory has focused on improving the limit of detection achieved with FIRE as a gas and liquid chromatography detector through an understanding of the limiting noise sources inherent in the system. This work has shown that with a simple FIRE detector the limiting noise is due to the residual flame background passed by the optical notch filter (5). In the case of the asymmetric stretching vibration of carbon dioxide at 4.4 pm, the flame background responsible for the limiting noise is due to a portion of an infrared emission band from water that partially overlaps the carbon dioxide band. If the flame background were perfectly stable, its effect could easily be eliminated by applying the appropriate dc offset to the input of the amplifier. However, when the background fluctuates, as it does with a flame, a simple dc offset cannot remove the fluctuations from the signal, and another approach must be used to reduce the noise arising from the background. In general, noise can be introduced into a measurement system as either an additive or a multiplicative component (7). To remove these unwanted signal components, the inverse mathematical operation must be performed (i.e., subtraction removes additive noise sources, while division removes multiplicative noise sources). Thus, to remove l / fnoise from a spectral sourse, source compensation (8) can be employed. However, to remove additive background, differential techniques are required. For signals that are subject to both types of noise, it is common practice to remove the additive background first. The two types of noise sources can be distinguished from one another by their effect on the signal. Since multiplicative noise sources modulate the signal, the noise amplitude from multiplicative noise sources will be directly proportional to the signal magnitude. By contrast, additive noise sources arise independently of the signal, and the noise amplitude from additive noise sources does not vary with signal strength. Since the flame background arises independently of the analyte signal, flame background fluctuations are an additive noise source, and their effect can be removed by simple subtraction using a differential amplifier. This paper describes a second-generation FIRE-GC detector that uses dual-beam optics to correct for additive background fluctuations in the source. In the dual-beam configuration, a beam splitter is used to divide the radiation from the source into two portions, each of which passes through different optical notch filters to select the bands associated with the appropriate background and the analyte. These filtered beams are then monitored by separate lead selenide detectors that form part of a Wheatstone bridge network whose output is
0003-2700/90/0362-1604$02.50/0C 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1. 1990
fed to the differential (A-B) input of a lock-in amplifier.
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EXPERIMENTAL SECTION Gas Chromatograph Interface. A gas chromatograph (Model
GC-BA, Shimadzu Scientific Instruments, Inc., Columbus, MD) with a thermal conductivity detector (TCD) and temperatureprogramming capability was used throughout this work. All separations were performed on a Carhopack-B (5% Fluorcol) packed column (Model 1-2425,Supelco, Inc., Bellafonte, PA). The outlet of the column was interfaced through the oven wall to the burner of the FIRE detector with a 12-cm-long, 600-pm4.d. stainless steel capillary tube (Model HTX-23, Small Parts, Inc., Miami, FL). To prevent analyte condensation, the stainless steel transfer tube was wrapped with heating tape (Part N-03122-30, Cole-Palmer Instrument Co., Chicago, IL) and maintained at 250 OC by power from a laboratory variac. Helium was used as a carrier gas and maintained at a flow rate of 30 mL/min. Analyte was introduced into the gas chromatograph by using conventional microsyringes (Model 701RN, Hamilton Co., Reno, NV). Flame Infrared Emission Detector. The premixed hydrogen-air. capillary-head burner was fahricated from aluminum stock and has been described previously (I, 4-6). The flow rates of hydrogen and air were controlled by means of standard gas flow meters (Part 3227-20 for hydrogen and Part 3227-26 for air, Cole-Palmer Co.), and the outlet pressures of the fuel and oxidant gases were regulated by triple-stage regulators as described previously (5,6). The optical and electronic components (excluding the lock-in amplifier and detector bias supply) of the dual-channel FIRE detector were mounted on a 10.0 X 5.0 X 1.0 em aluminum plate. The infrared emission from the h e was collected and collimated by a 5-cm, f / Z , CaF, lens (Part 43150, Oriel Corp., Stratford, CT). A 2.5-cm focal length, f / l concave mirror (Part 44350, Oriel Corp.), located at the Zf point behind the flame, was used to focus a unity magnified image of the flame onto itself. A light-beam chopper constructed from a 3000 rpm motor and a commercially available chopper blade (Part 220/10, Ithaco, Inc., Ithaca, NY) modulated the infrared radiation at 569 Hz. A 2.54-cm-diameter, 3.00-mm-thick ZnSe window (Part 4530, Oriel Corp.) served as an infrared beam splitter and was located at a position 45" to the normal of the incident collimated light heam. The collimated radiation in each of the channels of the FIRE detector was focused by a 5-cm focal length, f / 2 CaF, lens (Part 43150, Oriel Corp.) onto 1X 5 mm lead selenide detectors (Part P791, Hamamatsu Corp., Bridgewater, NJ). A 4.4-pm optical bandpass filter (Oriel Corp., Part 58300) with a full width at half-maximum (fwhm)of 0.15 pm was placed immediately in front of the PbSe detector in the analytical channel and was used to isolate the 4.42-pm COPemission band. A portion of the flame water emission band was isolated optically with a 3.00-pm bandpass filter (fwhm of 0.11 pm, Oriel Corp., Part 58160) positioned immediately in front of the PhSe detector in the reference channel. An aperture stop (Part ID-1.0, Newport Corp., Fountain Valley, CAI possessing a 2.5-cm maximum aperture was located in the reference channel and was used to balance the detector signals in the two channels optically. The lead selenide detectors were operated at room temperature and were biased from a regulated variable dc power supply (Part 6516, Hewlett Packard Corp., Avondale, PA). Precision resistors and capacitors (2% tolerance) were used throughout the preamplifier circuit. Two BiFET operational amplifiers (Part TL071, Texas Instruments, Dallas, TX) were employed as noninverting preamplifiers and both were powered from a standard regulated bipolar (*15 V dc) power supply (Part 2718, Heath Co., Benton Harbor, MI). The amplified outputs from the two operational amplifiers were subtracted and demodulated by a lock-in amplifier (Model 3962, Ithaco, Inc.) operated in the differentialinput mode. The lock-in amplifier employed a time constant of 1 s for all measurements. The output of the lock-in amplifier waz connected to a recorderlintegrator (Model 3394A, Hewlett Packard Corp.) after an appropriate attenuation by a standard voltage divider network. Reagents. All chemicals were chromatographic grade (Fisher Scientific Co., Pittsburgh, PA) and were used without further purification. Calibration standards of Freon-113 (CpC12FJwere prepared in dichloromethane to contain levels of 0.1-10 pg in 1
C"ROM1T0011AP"
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REFERENCE CHANNEL
Figure 1. Schematic diagram of the dual-channel FIRE detector for gas chromatography showing the gas chromat0graph:burner module (A). the optical rodule (B). and the elecboniclsignal processing module (C). Key: H,. hydrogen cylinder: Air, air cylinder; B. capillary burner; S. hydrogen-air flame source; C. light beam chopper; BmS. beam spliner; F,, 3.0-pm optical bandpass fiiter: F,. 4.4 pm optical bandpass filter;0, and D,. lead selenide detectors: PA, and PA,. preamplifiers; PSD, phase-sensitive detector; REC/INT. recorder-integrator. pL. The performance of the dual-channel FIRE detector was evaluated by using a laboratory-prepared Freon mixture. Procedure. Before use, the hydrogen-air flame of the FIRE detector was ignited, and the gas chromatograph and the FIRE detector were allowed to warm up for approximately 30 min. The phase angle of the lock-in amplifier was set in the single-ended input mode using the signal derived from the reference channel of the FIRE detector. After the phase angle had been set, the operation of the lock-in amplifier was returned to the differential input mode. RESULTS AND DISCUSSION Instrumental Configuration. A block diagram of the experimental arrangement for the dual-channel FIRE detector and gas chromatographic interface is shown in Figure 1. The dual-channel FIRE detector consists of three principal sections-the GC/burner module (Figure lA), the optical module (Figure lB), and the electronic signal-processing module (Figure 1C). The optical module collects the flame infrared emission from the miniature capillary-head burner and then divides the radiation into an analytical channel and a reference channel for detection. Radiation from each channel is detected and amplified by separate detectors and preamplifiers and then subtracted and demodulated by a single lock-in amplifier. As can he seen from Figure 2, infrared emission from the flame is collected and collimated by CaF, lens L. The collection mirror behind the capillary-head burner is used to increase the optical throughput by a factor of 2, and the mechanical chopper, C, is used to modulate the flame radiation. Following collimation of the radiation, the beam splitter (BmS, a standard ZnSe window) divides the radiation (reflected component, 30%; transmitted component, 70%) into two optical paths for focusing by the two CaF, lenses, L, and L,, onto detectors D, and D,, respectively. The optical handpass filter, F,, in the analytical channel is used to isolate the 4.4-pm CO, infrared emission hand. The optical bandpass filter, F,, (transmission notch centered at 3.0 pm) in the reference channel is used to isolate a portion of the 2.17-pm water infrared emission hand. The electronic signal processing system, shown in Figure 3, consists of three separate sections-the detector module (Figure 3A), the preamplifier module (Figure 3B), and the lock-in amplifier (Figure 3 0 . The two lead selenide detectors,
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ANALYTICAL CHEMISTRY, VOL. 62. NO. 15. AUGUST 1, 1990 ANALYTICAL CHANNEL
M
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Figure 2. Schemitic diagram of the optical module of the duaCchannel FIRE detector. Key: M. concave mirror: B. burner: F. H,-air flame: C, light beam chopper: L, L,. and L., calcium Ruwide lenses: BmS. zinc selenide beam spltter: AS. apertwe stop: F,. 3.0-lrm optical bandpass filter: F, 4.4-pm optical bandpass filter: D, and D,. lead selenide
detectors.
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Flgure 3. Schematic diagram of the electroniclsignal processing module of the dual-channel FIRE detector showing the Wheatstone bridge netw0& (A). the preamplifier circuits (E), and the lockin amplifier and readout assembly (C). Key: D, and D,. lead selenide detectors: R,. R,. and R,. bridge resistors: V. variable dc power supply: R,, feedback resistor: C,, feedback capacitor: PSD. phase-sensitive detector: RECIINT, recorder-integrator. Preamplifier gain control: R,. 300 kR; R,, 0: R,. 300 kR; R,, 0-20 kR: C,. 660 pF. Bridge balancing: R,. 0-200 kR; R,. 0 R,. 10 kR: R, 220 kR; C,. 10 pF. Optical attenuation: R,, 300 kR; R,. 0-20 kR: R,, 300 kR: R,. 220 kn; c,. i o
..
PF. D, and D, (corresponding to D, and D, in Figure 21, are operated at room temperature and are biased by the constant power supply VBB,through resistors R,, R,. and R,. (The function of resistors R,, R,. and R, will he discussed in the following section.) The PhSe detectors and biasing resistors form a Wheatstone bridge network. The preamplifiers consist of two ac-coupled voltage followers with gain. The preamplifier in the analytical channel has a fixed gain of 23, while the preamplifier in the reference channel has a variable gain (vide infra). The outputs of the preamplifiers are fed into the differential input (A-B) of the lock-in amplifier so that the demodulated output corresponds to the difference between the signal in the analytical channel and the signal in the reference channel. Background Subtraction Methods. During operation, the lock-in amplifier (LIA) output of the FIRE detector can
he adjusted to approximately zero by equalizing the differential mode inputs (i.e., equalizing the output signals from the two preamplifiers). This equalization should result in the rejection of unwanted signals that are common to both preamplifier channels. In the absence of an analytical signal, these common-mode signals may include additive flame background fluctuations that are wavelength independent as well as any common-mode ripple associated with the power supplies of the detectors or the preamplifiers. In optimizing the performance of the dual-channel FIRE detector, the goal in adjusting the differential input is to minimize signal fluctuations rather than adjusting the input of the LIA to zero. Once this background component has been minimized, no further adjustments need to he made, and the circuit can be operated in an unbalanced mode. Common-mode rejection of wavelength-independent, additive flame background fluctuations and power supply ripple can he accomplished in one of three ways. These include (1) adjusting the gain of the reference channel preamplifier until the signal fluctuations in the reference channel are equal to those in the analytical channel, (2) adjusting the bridge resistance (R, and R,) in the reference channel to balance the output of the Wheatstone bridge circuit, or (3) adjusting the light throughput of the reference optical path to match that in the analytical optical path. Preamplifier Gain Control Method. Because the lock-in amplifier performs real-time subtraction of the detector signals, one method that can he used for common-mode rejection of unwanted background fluctuations is to adjust the gain of the reference preamplifier so that the fluctuations in the demodulated output of the lock-in amplifier are minimized (Le., the background fluctuations in the reference channel are made approximately equal to those in the analytical channel). This gain control can easily he accomplished by adjusting the feedback resistance, Rf, of the reference preamplifier in Figure 3. It should be noted that, in the reference preamplifier gain control (RPG)method, the values of the bridge resistors (R, and RJ for both the reference and analytical channels are identical (within the tolerance of the components), Rzis zero, and the time constants of each preamplifier are adjusted to he nearly equivalent. Bridge Balancing Method. Another method of rejecting common-mode background fluctuations with the dual-channel FIRE detector is to balance the output of the Wheatstone bridge network in the absence of an analytical signal. This can he accomplished by varying the bridge resistance (R,) in the reference channel. In varying R, to balance the hridge, the range of variability must be limited so that the voltage drop across the lead selenide detectors does not fall below the minimum threshold for detection or exceed the maximum operating voltage. I t should be noted that with the bridge balancing (BB) method, Rz is zero, and the reference preamplifier feedback resistor, Rr, and capacitor, Cf, in Figure 3 are the same as those in the analytical preamplifier. Optical Attenuation Method. A third method for reducing unwanted, additive, common-mode background signals is attenuation of radiation in the reference optical path using an adjustable iris diaphragm (AS, Figure 2). With the optical attenuation (OA) method of background compensation, the magnitude of the radiance striking the reference detector is adjusted by closing down the aperture of the iris diaphragm until the fluctuations in the reference channel are approximately equal to the background fluctuations in the analytical channel. Fine-tuning of the reference signal can he accomplished by varying the resistor, R,. Common-Mode Noise Rejection. To remove background fluctuations from an analytical signal by subtraction, four prerequisites must he satisfied (1)The detection system must
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
not be detector-noise limited (i.e., the fluctuations must originate from a common source such as background emission or power supply ripple). (2) The background signal must be additive with respect to the analytical signal. (3) The background signal present in the analytical channel must be independently sampled by some means. (4) The sampled background fluctuations must be correlated with the background fluctuations present in the analytical channel. With the FIRE detector described previously ( 2 , 4 4 3 , the background radiance observed for the carbon dioxide band at 4.42 pm arises principally from spectral overlap with a portion of the 6.0-pm emission band due to the H-0-H bending mode of water. This background emission is particularly strong because the hydrogen-air flame produces large quantities of vibrationally excited water. While the combustion of hydrocarbon analytes also produces water, the volume of analyte present in the flame is relatively small. Therefore, the water produced from the analyte is insignificant compared with that produced by the flame, and the background radiance arising from the water emission of the flame is essentially independent of the signal arising from carbon dioxide. Thus, the background radiance and its associated noise should simply add to the COz radiance derived from the analyte. Because the hydrogen-air flame generates water as its principal combustion product, nearly all of the background infrared emission bands observed with the FIRE detector are due to water. Infrared emission from vibrationally excited water has been observed (3) as two bands centered at 2.77 and 6.00 pm. The water emission band at 6.00 pm extends from 4.0-10.0 pm, overlapping the COz emission band centered at 4.42 pm. While it should be possible, in principle, to compensate for background by monitoring the same water emission band that overlaps the analyte band (i.e., sampling in a spectral region removed from 4.42 pm, but located somewhere within the 4.0-10.0-pm region), this is not possible with the lead selenide detectors employed in this study. While these detectors provide excellent sensitivity at 4.42 pm, their long-wavelength response cutoff is at 5.5 pm. Since the COz emission band possesses a base-line width that extends from 4.1 to 5.0 pm, the combination of detector response and COz band width precludes the use of water emission in the region 4.0-10.0 pm. The second water emission band, centered at 2.77 pm, is due to H-0-H stretching. While this band does not overlap the COz emission band at 4.42 pm, it should still be suitable for background compensation provided the intensity fluctuations of the H-0-H stretching emission are correlated with those of the H-0-H bending emission, which fall within the bandpass of the optical notch filter used to isolate the COz emission. Since both water bands arise from the same molecule and both have approximately the same excitation potential, both should experience approximately the same excitation conditions in the flame. On this basis, it seemed reasonable to expect that intensity fluctuations for the two infrared emission bands of water would be correlated to some degree. Figure 4 shows a comparison of the background signal due to water emission that falls within the bandpass of the COz optical notch filter at 4.42 pm with the background signal arising from water emission at 3.0 pm. Both background signals in Figure 4 were obtained simultaneously by using identical instrument configurations with different optical bandpass filters. The dc component of both signals shown in Figure 4 has been removed, and the signals have been arranged directly above one another to facilitate comparison of time-dependent behavior without interference from absolute signal levels.
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Figure 4. Temporal correlation of the flame noise in the dual-channel FIRE detector showing the recorder tracing of the amplitude of the background fluctuation versus time in the reference channel (A, 3.0-pm optical bandpass filter) and the recorder tracing of the amplitude of background fluctuation versus time in the analytical channel (B, 4.4-pm optical bandpass filter).
It is readily apparent from Figure 4 that the time-dependent fluctuations in the water background within the bandpass of the optical notch filter at 4.42 pm are well correlated with those from the water background within the bandpass of the optical notch filter at 3.0 pm, even though the emissions arise from two different vibrational modes. Deviations from perfect correlation can be attributed largely to the detector noise (dark-current fluctuation) that arises from two independent sources and is, therefore, not expected to be correlated in the two channels. (Detector noise was measured by placing optical blocks immediately in front of both detectors and simultaneously recording the signals from the two channels. As expected, the signals were not correlated.) The flame background noise in the COz channel was found to be approximately 4.8 times that of the detector noise, while the flame background noise in the water channel (3.0 pm) was found to be approximately 3.5 times that of the detector noise. Thus, neither the analytical nor the reference channel is detectornoise limited, and the fluctuations in the flame background in both channels are highly correlated. On the basis of these results, subtraction of the background noise in the COzchannel using the real-time fluctuations in the water background at 3.0 pm is justified. Choice of Subtraction Mode. The three methods of background noise subtraction were evaluated in terms of their effectiveness in reducing background noise in the differential dual-channel FIRE detector, and the results were compared with the FIRE detector operating in the unsubtracted mode. The detection limit and signal-to-noise ratio (SNR) for Freon-113 (C2Cl3F3)were determined by using the different methods of background subtraction, and Table I summarizes the results. All entries in Table I are the result of five replicate 1-pL injections of Freon-113 chromatographed isothermally at 180 "C on a Carbopack-B (5% Fluorcol) column. Peak height was used as a measure of the signal. Noise levels were expanded to full scale for measurement, after which they were corrected mathematically to the same scale factor as the signal measurements. The detection limits are reported in ng and reflect a base-line elution time of 30 s. For all three background compensation methods, the phase angle between the output from the PbSe detectors and the
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Table I. Comparison of SNR Enhancement for the Three Different Modes of Background Subtraction
preamplifier gain
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120 0.15 800 131
121 0.53 228 468
115 0.06 1917 55
114 0.22 518 202
112 0.06 1867 56
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127 0.32 397 264 4.70
“Average of five replicates. bDefined at 2 times the rms noise. chopper reference circuit was adjusted by using the signal from the flame background (i.e., the water emission) in the single-ended input mode. The Ithaco Model 3962 lock-in amplifier, which sets the chopper-reference phase angle automatically, requires a strong signal to select the correct phase angle between the signal from the PbSe detector and chopper reference circuit. Since the goal of the differential FIRE detector is to minimize background noise and since this also reduces the magnitude of the background signal in the process, problems can arise with automatic phase setting of the lock-in amplifier in the differential mode when relatively weak signals are used. Using the relatively weak signal obtained after background subtraction to set the phase angle automatically can cause the amplifier to lock in on spurious signals and seriously degrade the SNR of the output. In optimizing the performance of the detection system, it was observed that as the signal from the PbSe detector decreased, the relative standard deviation in the phase angle set by the amplifier increased. In addition, it was also noted that the absolute phase angle set by the amplifier shifted to larger values as the input signal level decreased. These results indicate that at low input signal levels (such as occurs when background subtraction is employed), the lock-in amplifier erroneously sets the phase angle to incorrect values. Such incorrect phase settings could be due to the presence of spurious periodic signals in the system, although the actual source of any spurious periodic signals could not be identified. Thus, to maximize the SNR of the data acquired with the differential FIRE detector, the phase angle must be set with a strong detector signal such as that obtained in the singleended input mode. Table I shows that noise was reduced by a factor of 3-5 for all three background subtraction methods compared to the FIRE detector operating in the unsubtracted (single-ended) mode. [The differences in pairs of signal levels (subtracted mode and unsubtracted mode for each of the three methods) can be ascribed to irreproducibility in injection and difficulty in reproducing the three experiments exactly.] The noise reduction that results from background subtraction is reflected in an improvement in the SNRs and detection limits of the same magnitude (Le., a factor of 3-5). The minor differences in the improvements of the SNRs and detection limits for the three subtraction modes are probably not statistically significant. However, since the optical attenuation method is easier to implement, the remainder of this paper will develop and explore the ramifications of this method as representative of all three. Performance of the Optical Attenuation Method. The residual source noise observed after background subtraction by using the dual-channel FIRE detector was compared with the detector noise obtained by blocking the beams in both optical channels. The noise level obtained with both optical channels of the FIRE detector open (i.e., with flame background focused onto the PbSe detectors) is 1.4 times noisier than the noise level obtained with both optical channels blocked. Thus, even in the subtraction mode, the FIRE de-
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Figure 5. Plot of root-mean-square (rms) noise in the output of the subtracted mode as a function of the magnitude of the output signal.
tector is still source-noise limited. As stated previously, the optical attenuation method of performing background subtraction reduces the background noise by a factor of approximately 3-5. This results principally from a reduction in the additive noise from the water background lying within the bandpass of the C 0 2 optical notch filter. Thus, the noise output of the differential FIRE detector in the absence of analyte is, on the average, about 3-5 times lower than that obtained when the FIRE detector is operated in the unsubtracted mode. Therefore, on the basis of the comparison of the base-line noise reduction levels achieved by using the dual-beam FIRE detector, a factor of 3-5 would also be expected in SNR improvement. This value agrees quite well with the experimentally observed improvements in SNR reported in the preceding section. When the signals in the analytical and reference channels are balanced optically, the absolute signal level fluctuates around zero. Figure 5 shows a plot of the relative rootmean-square (rrns) noise as a function of the residual background voltage after subtraction. I t should be noted that (1) the background noise level is smallest when the difference between the signal levels in the two channels approaches zero and (2) the rms noise level approaches a constant value for residual background levels less than 1 mV. Optimization Studies. A series of optimization studies was conducted to determine the influence of flame conditions and detector supply voltage on the detection limit. For this purpose, Freon-113 was chosen as a test compound. Fuel-to-Oxidant Ratio. The effect of the hydrogen/air ratio on the detection limit was investigated by chromatographing a series of l-wL injections of Freon-113 isothermally at 180 “C.Figure 6 shows a plot of the fuel-to-oxidant ratio versus the detection limit for Freon-113 where each point represents the average of five replicate injections. Since air is only 20% oxygen, the optimum fuel-to-oxidant ratio of 1:1.4 corresponds to a fuel-rich flame. Detector Supply Voltage. The dual-channel FIRE detector, operated in the optical attenuation subtraction mode, shows a supply-voltage dependence with respect to the detection limit. Figure 7 shows this dependence by using the
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Hydrogen/Air Ratio Flgure 6. Plot of the detection limit for Freon-113 as a function of the hydrogen-air ratio as calculated from the volumetric flow rates of the gases. 120,
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Flgure 8. FIRE chromatograms for a 1-pL injection of a synthetic mixture consisting of (1) dichloromethane (309 pg), (2) trichlorofluoromethane (300 pg), (3) trichloromethane (330 pg), (4) trichlorotrifluoroethane (123 pg), (5) tetrachloromethane (300 pg), (6) hexane (33 pg), and (7) heptane (23 pg). (A) Chromatogram obtained in the dual-channel (subtracted) mode. (B) Chromatogram obtained in the single-channel (unsubtracted) mode. Conditions: Carbopack-B (5 % Fluorcol) packed column; He carrier gas (30 mL min-I); linear temperature program from 160 to 220 OC at 10 OC min-I.
Detector Supply Voltage (V) Flgure 7. Plot of the detection limit for Freon-113 as a function of detector supply voltage.
detection limit for Freon-113 as an example. Each point on the plot represents an average of five replicate injections of 1 pL of Freon-113, chromatographed isothermally at 180 "C. From Figure 7 , the optimum supply voltage for the PbSe detectors and the optical configuration used in this study was about 65 V. Applications to Chromatography. Before applying the dual-channel FIRE detector to gas chromatography, its linearity and dynamic range were studied. Using Freon-113 as a test compound, it was found that the dual-channel FIRE detector possesses the same high degree of linearity that the single-channel FIRE detector exhibits ( 4 ) and that the improved signal-to-noise ratio obtained with the dual-channel FIRE detector further extends the linear dynamic range at low concentrations by about a half a decade of concentration (from about 4.0 to about 4.5). Some curvature, observed at the high concentration limit, could be due to self-absorption, incomplete combustion, or a combination of the two. Under actual chromatographic conditions, the dual-channel FIRE detector significantly reduces background noise in comparison to the single-channel FIRE detector operating in the unsubtracted mode. Figure 8 shows comparison chromatograms for a mixture of Freons and hydrocarbons obtained with a dual-channel FIRE detector and a single-channel FIRE detector. Each chromatogram was obtained from a 1-pL injection of test sample under identical chromatographic conditions. As expected from the foregoing discussion, Figure 8 shows that the SNRS of all chromatographic peaks have been improved by a factor of approximately 3.6 when the subtracted mode of operation is compared with the unsubtracted mode. For comparison, Figure 9 shows the chromatograms obtained from 1-pL injections of test sample using the dualchannel FIRE detector and a commercially available thermal conductivity detector (TCD). Both chromatograms in Figure 9 were obtained under the same chromatographic conditions.
0
2
4
6
8
Retention Time (min) Figure 9. Chromatograms for a 1-pL injection of a synthetic mixture consisting of (1) dichloromethane (129 pg), (2) trichlorofluoromethane (219 pg), (3) trichloromethane (190 pg), (4) trichlorotriiuoroethane (232 pg), (5) tetrachloromethane (202 pg), (6) hexane (121 pg), and (7) heptane (125 pg). (A) Chromatogram obtained with a thermal conductivity detector. (8)Chromatogram obtained with the FIRE detector in the dual-channel mode. Conditions: Carbopack-B (5 % Fluorcol) packed column; He carrier gas (30 mL min-'); linear temperature program from 160 to 220 O C at 16 OC min-'.
On a purely qualitative basis, Figure 9 indicates that the dual-channel FIRE detector has a sensitivity nearly equal to that of the TCD. To provide a more quantitative comparison, the mass flow rate detection limit for Freon-113 was converted into the equivalent concentration detection limit on a volume basis. From the mass flow rate detection limit obtained from Freon-113 with the dual-beam system (56 ng/s) and the carrier gas flow rate (30 mL/min), the concentration of Freon-113 in the carrier gas at the detection limit corresponds to 15 ppm on a volume basis. Since detection limits with the thermal conductivity detector are typically in the range 10-100 ppm
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Anal. Chem. 1990, 62, 1610-1615
(9),it is clear that the performance of the dual-channel FIRE detector is comparable to that of a typical TCD. Examination of Figure 9 also shows that the response of the dual-channel FIRE detector probably becomes greater than that of the TCD detector when the number of carbon atoms in the analyte increases (i.e., for hexane and heptane). This increase in response for the FIRE detector undoubtedly reflects an increase in the number of COz molecules produced by combustion of these higher molecular weight analytes in the hydrogen-air flame.
CONCLUSIONS This paper describes a new dual-channel FIRE detector for gas chromatography that reduces the adverse effects of additive background noise in the detector output by subtracting both the dc component of the background and the commonmode background fluctuations from the analytical channel. Because the dc component of the flame background is removed by the dual-channel instrument, the lock-in amplifier can be used at higher gain settings than were possible in the single-channel (unsubtracted) mode. Using the dual-channel configuration, detection limits for Freon-113 are improved by a factor of about 3-5 compared with a single-channel FIRE detector. At its present stage of development, the flame
infrared emission GC detector is about as sensitive as a TCD detector. Registry No. HOH, 7732-18-5.
LITERATURE CITED Busch, K. W.; Busch, M. A.; Tilotta, D. C.; Kubala, S. W.; Lam, C. K. Y.; Srinivasan, R. Spectroscopy 1989, 4(8), 22-36. Hudson, M. K.; Busch, K. W. Anal. Chem. 1989, 59. 2603-2609. Tilotta, D. C.; Busch, K. W.; Busch, M. A. Appl. Specfrosc. 1989, 43,
704-709. Hudson, M. K.; Busch, K. W. Anal. Chem. 1988, 60, 2110-2115. Kubaia, S. W.; Tilotta, D. C.; Busch, M. A,; Busch, K. W, Anal. Chem. 1989, 6 1 , 1841-1846. Kubah, S. W.; Tilotta. D. C.; Busch, M. A,; Busch, K. W. Anal. Chem . 1989, 61, 2785-2791. Hieftje, G. M. Am. Lab. 1988, 20(10), 110, 112-115. Busch, K. W.; Busch, M. A. MulHelement Detection Systems for Spectrochemical Analvsis ; Wiley-Interscience: New York, 1990; Chapter 13, p 529. Strobel, Howard A.; Heineman, William R. Chemical Instrumntetion: A Systemtic Approach; Wiley Interscience: New York, 1989; Chapter 25, D 917.
RECEIVED for review January 23,1990. Accepted May 7,1990. This work was supported by Baylor University Research Grants 012-S85-URC, 006-S87-URC, 007-F87-URC, and 0100-032-1510Infrared Emission Research.
Derivatized Cyclodextrins for Normal-Phase Liquid Chromatographic Separation of Enantiomers Daniel W. Armstrong,* Apryll M. Stalcup, Martha L. Hilton, Jo Dee Duncan, James R. Faulkner, Jr., and San-Chun Chang
Department of Chemistry, University of Missouri-Rolla, Rolla, Missouri 65401
Several different derlvatired fi-cyclodextrins were synthesized and used as chirai statlonary phases In normal-phase liquid chromatography. The multlply substituted derivatives were made wlth acetic anhydride, ( R ) - and (S)-1-( 1-naphthyl)ethyi Isocyanate, 2,6-dlmethylphenyi Isocyanate, and p-toluoyl chloride. The flrst successful cyciodextrln-based, normaiphase separation of enantlomers was accomplished on these derivatlve phases. I n contrast to chirai separations on the natlve jhyclodextrin stationary phase, the enantiomeric separation mechanism on these new phases Is not thought to be dependent on lnciuslon complexation. The sbnllaritles and differences between the derlvatized cyclodextrin statlonary phases and the cellulosic statlonary phases are dlscussed.
INTRODUCTION Cyclodextrin bonded phases have been used for the reversed-phase separation of a variety of enantiomers (I),diastereoisomers (Z ) , structural isomers ( 2 ) ,enzymes (3), and routine compounds (4). Of these, the enantiomeric separations probably have received the greatest attention. In order to evaluate the mechanism of enantioselective chromatography, a number of empirical and theoretical studies have been done (5-9). In specific cases involving cyclodextrins, the formation of an inclusion complex seems to be a fundamental part of the chiral recognition and separation process ( 1 , 2 , 4 , 9). As yet, there have been no reports of normal-phase, enantiomeric
separations on cyclodextrin bonded phase columns. Indeed, the fact that facile enantiomeric resolution obtained in the reversed-phase mode could not be duplicated in the normal-phase mode has been used as indirect evidence to support the premise that inclusion complexation is necessary for enantioselectivity (9). However, cyclodextrin bonded phases have been used successfully in normal-phase liquid chromatography (LC) for a number of achiral separations (10-12). The retention behavior was somewhat like that of a diol column. It is believed that the nonpolar portion of the mobile phase (e.g., hexane, heptane, etc.) occupies the cavity of cyclodextrin and that solute retention was due mainly to interaction with the external hydroxyl groups that line the top and bottom of the cyclodextrin torous (10-13). While it is interesting that native cyclodextrins (i.e., cyclic a-l,klinked glucose) do not seem to effectively resolve enantiomers under normal phase conditions, there are analogous examples for other naturally occurring chiral molecules. For instance, cellulose (linear /3-1,4 linked glucose) seems to be much more effective as a chiral stationary phase when extensively derivatized. Triacetyl cellulose was one of the first derivatives to be used in this way (13). Subsequently, a large number of aromatic cellulose derivatives were shown to be even more widely useful (14). Typically, all of the derivatized cellulose columns are used in the normal-phase mode. The possibility that derivatized cyclodextrin bonded phases could be used for enantiomeric LC separations in the normal-phase mode has not been considered previously. If derivatized cyclodextrins have good enantioselectivity under
0003-2700/90/0362-16 10$02.50/0 0 1990 American Chemical Society
