Anal. Chem. 1980, 52, 897-901
897
Chemiluminescent Aerosol Spray Detector for Liquid Chromatography John W. Birks' and Myron C. Kuge Department of Chemistry and Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, Colorado 80309
A detector for liquid chromatography (LC) based on the observation of ozone-induced chemiluminescence (CL) is described. The LC effluent is nebulized by a high velocity stream of 03/0,gas. The CL produced is detected by a photomultlplier tube having an extended red response using the photon counting method. The chemiluminescent aerosol spray detector responds selectively to highly fluorescent compounds, olefins, divalent sulfur compounds, and certain nitrogen compounds such as hydrazines, azides, and nkrogen heterocycles. It is likely that additional selectivity may be achieved by making use of optical filters for wavelength discrimination. Detection limits are in the low microgram to low picogram range. The detector approximates a mass detector, the integrated response being only weakly dependent on the liquid flow rate. The detector may be used with nearly all LC solvents.
In those cases where the phenomenon of chemiluminescence (CL) has been applied t o chemical analysis, the method has generally proved t o be extremely sensitive and selective. In the gas phase, CL is the method of choice for the determination of ozone and nitric oxide in air using the NO + O3 chemiluminescent reaction ( I ) . A gas chromatography (GC) detector based on the CL produced upon reaction of the GC effluent with ozone has recently been described and shown t o be selective for olefins ( 2 ) . CL detectors for gas chromatography based on reaction of the GC effluent with fluorine atoms t o achieve iodine selectivity (3)and nitrogen atoms (4) for universal detection have recently been reported. Nitrosamines are frequently detected using a novel CL detector in which the N-NO bond is cleaved by pyrolysis, followed by reaction of the resulting nitric oxide with ozone (5-10). This detector may be used for either gas chromatography ( 1 1 ) or liquid chromatography (12). In aqueous solution, the metal catalyzed CL reaction of oxidizing agents with luminol has been used for the determination of oxidizing agents such as HzOz(13),C10- (14), C12 ( 1 5 ) ,and I2 (16) and for the determination of a variety of metal ions such as Fez+ ( I n ,CuZf (18), and Cr3+ (19). The CL oxidation of gallic acid has been used t o determine trace concentrations of formaldehyde and other organics (ZO), and t o determine trace concentrations of Co2+ and a few other metal ions (21). We describe here a new detector for liquid chromatography (LC) based on chemiluminescence produced when the LC effluent is nebulized by a high velocity stream of 03/02 gas. This chemiluminescent aerosol spray (CLAS) detector may be used with a variety of solvents commonly employed in liquid chromatography, and has been found t o be selective for certain nitrogen and sulfur compounds, alkenes, and some highly fluorescent compounds with detection limits at the low microgram t o low picogram level.
EXPERIMENTAL Detector Design. The greatest difficulty encountered in the design of the CLAS detector was that of memory. The use of a 0003-2700/80/0352-0897$01 .OO/O
nebulizer to produce a fine spray of the sample solution is an idea borrowed from atomic flame spectrometry. A flame may be operated unconfined since it vaporizes the sample injected into it, but a CLAS detector must be enclosed in order to contain and remove the liquid waste. For large reaction cells, the aerosol evaporates on the surface of the cell, leaving behind the less volatile components which continue to react with ozone a t the surface and produce light. The solute is very slowly removed by reaction, resulting in a very large memory of the order of several minutes to several hours. Our spray detector eliminates this problem by using a small reaction cell in which the interior surface is continuously washed down by the spray itself. A schematic of the detector is provided as Figure 1. Essentially, the detector consists of a nebulizer (A), a reaction cell (B) that resembles an inverted test tube, and a light detection system. A fine spray of the LC effluent is produced by a high velocity stream of the 03/02 gas and directed toward the top of the reaction cell (bottom of the inverted test tube). The aerosol strikes the surface where a liquid film is formed which subsequently is removed by the influences of both gravity and the flow of gas. Light may be produced either in the liquid phase, gas phase, or a t the gas/solution interface owing to chemical reaction and energy transfer processes. The light is reflected by a rhodium-coated ellipsoidal mirror ((C), Melles Griot 02-REM-001) onto a photomultiplier tube (PMT, (D)). To efficiently collect the light, one focus of the ellipsoid is placed inside the reaction cell, and one focus is placed on the photocathode. The reaction cell is externally painted flat black except for a narrow band (E) above the nebulizer of length 0.5 cm (the detection zone), thus blocking room light and limiting the detection volume. The nebulizer consists of a 2.&mm o.d., 1.2-mm i.d. Pyrex tube (A) into which is fed a length of loosely fitting flexible polyethylene capillary tube ((F),Fisher 14-170-llB) of the type commonly used in the nebulizers of atomic absorption spectrophotometers. The nebulizer passes through the reaction cell wall via a glass blown ring seal and is enlarged to 6-mm 0.d. external to the reaction cell for coupling to a '/,-inch Swagelok union tee (G) using Teflon ferrules. The reaction cell is held in place and adjusted vertically for maximum sensitivity by a 3/,-inch O-ring compression fitting ((H),Cajon Ultra-Torr). A vacuum valve (I) allows the chamber containing the mirror and reaction cell to be evacuated and sealed to prevent frosting of the cell window due to cooling caused by the adiabatic expansion of gas a t the nebulizer. The 03/02 gas enters the bottom of the union tee (G). A short piece of '/,-inch 0.d. tubing connects the union tee to a Swagelok '/4-inch to 1/16-inch reducing union (J). The polyethylene tube carrying the liquid passes through the top of the tee and through the reducing union. It is sealed to the '/,,-inch connector using Teflon ferrules and a collar of '/16-inch 0.d. polyethylene tubing. The entire detector housing is attached via O-ring seals to an EM1 FACT-50 thermoelectrically cooled PMT housing containing an EM1 9658RA PMT. The PMT is wired with the anode at ground and the photocathode a t -1500 V. This PMT has an extended red response. The short wavelength response is limited to -310 nm by the window material of the PMT and detector as well as by the absorption of ozone. The signal from the PMT was detected by photon counting using an ORTEC model 454 pulse amplifier, model 346 discriminator, and model 9315 counter. Apparatus. A schematic diagram of the entire experimental system is provided as Figure 2. The CLAS detector may be used either to aspirate the sample directly from a beaker or it may be attached to a liquid chromatograph. For these studies, a Spec1980 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 5 2 , NO. 6, M A Y 1980
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Figure 3. Effect of gas and liquid flow rates on the detector response for 10-pL injections of 1.5 ppm Rhodamine B in methanol. Liquid flow rate of 1.0 mL min-'. Gas flow rate of 4.0 STP L min-' F
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Figure 1. Schematic diagram of the CLAS detector r----
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Figure 2. Schematic of the complete measurement system, including the liquid chromatograph
tra-Physics model 3100 L C was used with a loop injector (HPSV-20). This allowed a continuous flow of solveni through the detector. Elution of an LC peak was simulated by interruption of the solvent flow with a 10-pL injection of sample solution. For most experiments, the signal was counted for a 30-s interval following injection of the sample and subsequently corrected for the blank. Time-resolved experiments utilized a Tracor Northern model TN-1505 signal analyzer for storage of photon counts in 2048 channels. The memory of the signal analyzer was output to an X-Y recorder as a permanent record. Reagents. Most chemicals studied were selected from a collection of compounds obtained from Chem Service, Inc., West Chester, Pa. All solvents were ACS certified. Sources of compounds are identified in the tables presented. Oxygen was Industrial (welding) grade, obtained from General Air Service and Supply, Denver, Colo. Ozone was produced by electrical discharge of molecular oxygen using an ozonizer of our own construction, but similar to those available commercially. The fractional conversion of molecular oxygen to ozone was typically 2 70,as determined by the standard iodometric method, but varied with the oxygen flow rate.
RESULTS A N D D I S C U S S I O N Gas a n d Liquid Flow Rate Dependence. The effects o f gas and liquid flow rates on the detector response arc illus-
Figure 4. Time-resolved response of the CLAS detector to a 10-pL iniection of 100 ppm Rhodamine B
trated in Figure 3. This figure shows the integrated response of the detector to 10-pL injections of 1.5 ppm Rhodamine B in methanol. As can be seen in the figure, there is an optimized Q 3 / Q 2 flow rate for this particular detector near 4.0 STP L inin-'. T h e effect of liquid flow rate on the integrated response between 0.5 and 1.6 mL min-' is not large. A sharp decrease in response for liquid flow rates below 0.5 mL min-' was observed. This effect is largest for the lowest gas flow rate, and is probably due to a failure of the liquid to be efficiently nebulized at low liquid flow rates. The weak dependence of the integrated signal on liquid flow rate suggests that. the detector approximates a mass detector rather than a concentration detector. T o test this hypothesis, a sohtion of Rhodamine B was continuously introduced to the detector by filling the solvent reservoir of the LC with a 1.5 ppm solution. The count rate was found t o increase linearly with increasing liquid flow rate over the range 0.5 to 1.5 mL/min, thus confirming that the detector approximates a mass detector over a limited range of flow conditions. For these conditions, the mass of analyte, ma,in the detection zone is given by where c, is the analyte concentration, E;, is the liquid flow rate, and T i? the residence time in the detection zone (i.e., the reciprocal of the exponential decay constant for removal of liquid from the detection zone). As will be seen below, T is a function of the gas flow rate. Time-Resolved Response t o a 1O-wL Injection. The rime-resolved response to a IO-& plug injection of 100 ppm Rhodamine B in methanol is shown in Figure 4 for a liquid flow rate of 1.0 m L min-' and the optimized gas flow rate of
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
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CONCENTRATION Figure 5. Working curve for Rhodamine 6 for aspiration of solutions. G a s flow rate of 7.0 STP L min-' and a resulting liquid flow rate of 2.0 mL min-'
4.0 STP L min-'. The leading edge of the plug of sample reaches the detector in 3.4 s, and the maximum response is reached after an additional 1.6 s. T h e decay of the signal occurs more slowly and is approximately exponential with a 7 s). This corresponds to a decay constant of 0.14 s-l ( T continuous dilution of a detector volume of -120 pL. After 30 s, the signal has returned to within 1% of base line. Higher gas flow rates were found to produce narrower time-resolved responses and thus smaller detector dead volumes, but at the expense of reduced integrated signals. This effect is responsible for the reduction in integrated signal by about a factor of two when the gas flow rate is increased from 4.0 to 5.5 STP L min-' (Figure 3). W o r k i n g C u r v e f o r Aspirated Sample. Figure 5 is the working curve obtained upon aspirating samples of Rhodamine B in methanol. In order to aspirate samples, a higher gas flow rate than that determined to be optimum for injected samples must be used. For this working curve, an 03/02 flow rate of 7.0 STP L min-' was used. This resulted in a liquid flow rate of 2.0 mL min-'. The curve is linear over the three orders of magnitude between 15 ppb and 15 ppm (slope = 1.05). There is negative curvature between 15 ppm and 150 ppm (slope = 0.45). A likely cause of the curvature a t high concentrations is absorption of a fraction of the chemiluminescence by the Rhodamine B which is visibly colored a t the higher concentrations. The aspiration feature of the CLAS detector is useful for the analysis of samples when no separation step is required. W o r k i n g C u r v e s f o r I n j e c t e d Samples. The working curves for four compounds based on the integrated responses t o 10-pL injections of the samples dilute in methanol are provided in Figure 6. All of these data were obtained using a liquid flow rate of 1.0 mL min-' and an optimized 03/02 gas flow rate of 4.0 STP L min-l. Rhodamine B again shows linearity over three orders of magnitude; in this case, between and g absolute. Methylene Blue displayed negative curvature over the four orders of magnitude tested. Since all of the solutions were highly colored, this is likely due to absorption of a portion of the CL produced. The two nitrogen heterocycles, quinoline and indole, provide quite different responses. The working curve for quinoline is linear over three orders of magnitude (slope = 0.95), whereas the indole curve is supralinear. Note t h a t the sensitivities to quinoline and indole are three t o four orders of magnitude less than for Methylene Blue and Rhodamine B. A working curve for tannic acid was found to be linear (slope = 0.97) over the range tested, 0.5 to 50 pg. U s e f u l Solvents. In Table I the integrated responses to 10-pL injections of pure solvents are tabulated, the carrier liquid being methanol. No blank corrections were made to
Figure 6. Working curves for Methylene Blue, Rhodamine B, quinoline, and indole based on int rated responses to 10-yL injections. Gas flow rate of 4.0 STP L min . Liquid flow rate of 1.0 mL min-'
9
Table I. Response of CLAS Detector t o Common Solvents and Effect of Solvents o n CL Intensity
solventa aniline acetonitrile carbon tetrachloride chloroform cyclohexane cyclohexene hexanes (isomeric mixture) methanol nitromet hane pyridine
tetrahydrofuran toluene water (distilled)
pure solvent, counts x 10-3 38 800 1120 136 143 140 7 7 282 140
160 158 37 2 248 143 147
1 PPm Rhodamine B in solvent, counts X 10-3
3 460 789 2 120 2 490 3 360 1920 1180 1 4 200 4 760 3 270 1800
a All organic solvents were obtained from Fisher except for cyclohexene which was obtained from Eastman.
these data. Except for cyclohexane, these are commonly used solvents in liquid chromatography. I t may be concluded from Table I t h a t acetonitrile, carbon tetrachloride, chloroform, cyclohexane, hexane, methanol, nitromethane, pyridine, tetrahydrofuran, toluene, and water are all useful solvents for the CLAS detector. The third column of Table I provides the integrated responses to 10-pL injections of 1ppm Rhodamine B in the various solvents. These counts have been corrected using the data in the second column as the blank. Some solvents are seen to provide enhancement of response over that for pure methanol. In particular, pyridine enhanced the signal by a factor of 7.4 and tetrahydrofuran enhanced the signal by a factor of 2.5. I t should be noted that in these experiments the principal solvent is still methanol since the injection volume is only 10 pL of the total detector dead volume of 120 pL. Sensitivities a n d Detection L i m i t s f o r a Variety of O r g a n i c Compounds. The sensitivities to 60 organic compounds were determined by making 10-pL injections of either 1%or 10 ppm solutions in methanol. All solutions were 1% except Methylene Blue, Rhodamine B, fluorescein, 4,5-dibromofluorescein, 2,4,5,7-tetraiodofluorescein,2,4,5,7-tetrabromofluorescein, phenolphthalein, Methyl Orange, Methyl Red, Eriochrome Black T , and Celanthrene Fast Blue FFS which were 10 ppm. Detection limits were computed assuming linear working curves as follows. First, the standard deviation of the blank was determined from 50 thirty-second background
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
-___ Table 11.
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_
_
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~
Detection Limits for Compounds Tested detection sourceQ limit, p g compound
A. Polyaromatics 1. naphthalene 2 . anthracene'
100
