Anal. Chem. 2001, 73, 1387-1392
A Sensitive Small-Volume UV/Vis Flow Cell and Total Absorbance Detection System for Micro-HPLC Mauro Aiello and Robert McLaren*
Department of Chemistry and Centre for Atmospheric Chemistry, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3
An optical multichannel absorbance detection system suitable for micro-HPLC is described. The detection system includes a flow cell with detection volume of 30 nL and path length of 12 mm. A fiber optic spectrometer with charge-coupled device array is used to collect spectral information at 1 Hz frequency. Signal-to-noise ratios are enhanced through the use of large bandwidth total absorbance signals, while linearity and spectral resolution are maintained. Theoretical predictions of bandwidth dependent signal-to-noise ratios of the total absorbance signal are compared to experimental observations, showing that optimum total absorbance signal bandwidths occur at 2.8 σabs for a Gaussian absorption band under light noise limited conditions. This bandwidth is much larger than bandwidths used in most commercial detectors. The detection system is applied to the separation of 2,4-dinitophenylhydrazones of organic carbonyls sampled from tropospheric air samples. For these hydrazones, a bandwidth of 181 nm is used for detection and calibration, which results in more universal hydrazone detection. The analytical separations field has seen tremendous advancements in the past decade including the commercialization of capillary electrophoresis1 and advances toward the routine use of capillary electrochromatography.2 At the same time, advances in the more conventional analytical separation methods such as highperformance liquid chromatography (HPLC) have been in the direction of miniaturization, in terms of both column sizes and the size of packing materials. Micro-HPLC has been defined in one nomenclature as chromatography with columns of inner diameter 100 µm < dc e 1000 µm and its advantages have been outlined previously.3 These advantages include the economics of significantly reduced solvent consumption and the ability to analyze smaller samples without degradation of concentration detection limits. To illustrate the former advantage, a typical flow rate for a separation using standard analytical (4.6-mm i.d.) columns is 1000 µL/min whereas the flow rates necessary to achieve the same linear velocity and separation using 1.0, 0.3, or (1) Baker, D. R. Capillary Electrophoresis; John Wiley and Sons: New York, 1995. (2) Dadoo, R.; Yan C.; Zare, R. N.; Anex, D. S.; Rakestraw, D. J.; Hux, G. A. LC-GC 1997, 15, 630-5. (3) Krejci, M. Trace Analysis with Microcolumn Liquid Chromatography; Marcel Dekker: New York, 1992. 10.1021/ac000943e CCC: $20.00 Published on Web 02/17/2001
© 2001 American Chemical Society
0.1-mm-i.d. columns is only 50, 5, and 0.5 µL/min, respectively. These flow rates are predicted by a scaling factor, fscale.
fscale ) [dmicro/danalytical]2
(1)
where dmicro is the internal diameter of the micro column and danalytical is the internal diameter of a conventional analytical column. To maintain the efficacy of the separation with smaller diameter columns, the detection volume must be reduced. One can predict the required detector volumes for microcolumns by using the same scaling factors. Alternatively, one can predict the detection volume using the smallest peak volume one expects to encounter in the separation. For example, an analyte peak eluted from a goodquality HPLC column with 20 000 theoretical plates with a retention time of 10 min has a full width at half-maximum (fwhm) of 0.166 min. The peak volume is given by multiplying the peak width by the flow rate. To ensure that the detector does not degrade the resolution, the detection volume should be at least 20 times smaller than the peak volume. Using flow rates discussed above, the corresponding detection volumes should be 8.3 µL, 416 nL, 42 nL and 4.2 nL for 4.6-, 1.0-, 0.3-, and 0.1-mm columns, respectively. The most desirable approach to achieving low detection volumes is on-column detection.4 For the lowest volumes listed above, this is possible using techniques such as laser-based fluorescence5 and thermooptical methods.6 Unfortunately, fluorescence detection is limited to a small class of compounds and on-column thermooptical methods in general do not easily yield spectral information to aid in qualitative identification of analytes. Ultraviolet/visible absorption is still one of the most common detectors used in HPLC. Historical advances in UV/vis detectors followed the development of fixed-wavelength detectors, dualwavelength detectors, variable-wavelength detectors, and finally optical multichannel devices.7 A reduction in detector volume for UV/vis detectors usually implies a reduction in path length and, hence, a reduction in sensitivity. Reducing the aperture (crosssectional area) of the flow cell while maintaining the path length (4) Yang, F. J. J. High Resolut. Chromatogr. Chromatogr. Commun. 1981, 4, 83-5. (5) Dovichi, N. J. Rev. Sci. Instrum. 1990, 61, 3653-67. (6) Nolan, T. G.; Bornhop, D. J.; Dovichi, N. J. J. Chromatogr. 1987, 384, 18995. (7) Stevenson, R. L. In Liquid chromatography detectors; Vickrey, T. M., Ed.; Marcel Dekker: New York, 1983.
Analytical Chemistry, Vol. 73, No. 6, March 15, 2001 1387
Figure 1. Schematic diagram of the microflow cell with path length of 12 mm and detection volume of 30 nL.
is a possible approach to reduce detection volumes, although this is sometimes discouraged due to shot noise limitations.8 Previous approaches to the development of small-volume, high-sensitivity absorbance flow cells for use in micro-HPLC and capillary electrophoresis have included axial propagation of light through capillaries with “Z”- and “U”-shaped capillary cells,9,10 the use of small flow cells with square silica channels,11 rectangular channels,12 multireflection cells,13 and a unique double eccentric bend capillary flow cell.14 These cells all have large values of the path length-to-aperture ratio,10 such that light throughput can be a limiting factor for the absorbance detection limit. In this paper, we describe a simple but sensitive small-volume UV/vis flow cell with 12-mm path length and 30-nL detection volume. The flow cell is coupled to the detection system via fiber optics. The optical measurement system is a fiber-optic CCD spectrometer. To overcome shot noise limitations, signal-to-noise (S/N) ratios are increased by calculating what we refer to as the total absorbance signal (TAS). The TAS is a large spectral bandwidth absorbance measurement that maintains signal linearity that is inherent in Beer’s law, as well as spectral fidelity that can be important for peak identification.15 The TAS detection scheme can be used with diode array or charge-coupled device detectors to increase absorbance S/N ratios, especially for detectors where light throughput is a limiting factor. While selectivity is sacrificed with a large spectral bandwidth measurement, the option for selectivity is retained if the spectral information from which the TAS is calculated is stored for recovery. Theoretical and experimental TAS signal-to-noise ratios are compared. In total, the detection system described in this paper is a sensitive but (8) Riordon, James R. Anal. Chem. 2000, 483, 483A-7A. (9) Chevret, J. P.; van Soest, R. E. J.; Ursem, M. J. Chromatogr. 1991, 543, 439-49. (10) Moring, S. E.; Reel, R. T.; van Soest, R. E. J. Anal. Chem. 1993, 65, 34549. (11) Hewlett-Packard, High Sensitivity Detection Cell for HP 3D Capillary Electrophoresis System; 12-5965-5984E, Boeblingen, Germany, 1997. (12) Tsuda, T.; Sweedler, J. V.; Zare, R. N. Anal. Chem. 1990, 62, 2149-52. (13) Wang, T.; Aiken, J. H.; Huie, C. W.; Hartwick, R. A. Anal. Chem. 1991, 63, 1372-6. (14) Abbas, A. A.; Shelly, D. C. Anal. Chim. Acta 1999, 397, 191-205. (15) Hewlett-Packard, Diode-array detection in capillary electrophoresis-Part 2: Spectral Fidelity, Peak Purity, and Library Searching; 12-5963-1891E, Boeblingen, Germany, 1994.
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inexpensive optical multichannel micro-HPLC detector. This detection system has been in use as part of a dedicated sampling and analysis instrument for the measurement of organic aldehydes and ketones in tropospheric air.16 EXPERIMENTAL SECTION The flow cell design is shown in Figure 1. The cell was made from two blocks of stainless steel. The mated sides of the stainless steel were polished to a smooth surface to aid in sealing. A smalldepth optical channel, 12 mm long, was machined into one of the mated sides by using a high-speed steel tool. Connecting tubing (1/16 in. o.d. × 0.005 in. i.d. PEEK) was epoxied into two 1/16-in. holes drilled into the edges of the top block that connected at their bottom end with the optical channel. The ends of the optical channel were sealed with 2-mm-thick quartz windows. The final optical channel was triangular in shape with ∼50-µm depth, as measured with an optical microscope. The volume of the detection region is ∼30 nL with a path length of 12 mm. Light from a 30-W deuterium lamp (Oriel) was delivered to, and collected from, the flow cell using 400-µm fiber optics. The end of the fiber optic was mounted such that it was touching the quartz window directly in line with the optical channel. The same was done on the collection end except that the fiber was mounted on an x-y-z translation stage for optimizing the position of light collection. The transmitted light was coupled into a miniature fiberoptic spectrometer (Ocean Optics, S2000) with a 2048-element linear CCD detector, 600 lines/mm grating blazed at 400 nm, and 100-µm slit. The optical resolution of the spectrometer is ∼4 nm (fwhm). The CCD pixel resolution is 0.38 nm/pixel element The µ-HPLC system consisted of two micropumps, mixer, and controller (Micro-Tech Scientific), and a 1.0 × 250 mm C-18 column (3.5 mm Zorbax). Total flow rates were 50 µL/min. The two solvents were (A) 100% H2O and (B) 100% acetonitrile. The solvent program was as follows: 80% A/20% B at 0 min, increased linearly to 100% B at 60 min. For collection of air samples, a custom sampling cartridge was made from stainless steel, 30-mm length, 1-mm i.d., packed with 40/60 mesh silica gel. A 2,4-dinitrophenylhydrazine (DNPH) (16) Aiello, M.; McLaren, R. Proc. Enviro-Analysis ‘2000, Ottawa, 2000; pp 2138.
derivatization solution was made by adding 0.3 g of recrystalized DNPH and 10 µL of concentrated H2SO4 to a 50-mL volumetric flask and filling with 95% acetonitrile (ACN)/5% water. This solution was constantly maintained under ultrahigh-purity helium at 50 psi to maintain purity and isolate it from atmospheric air. Immediately before sampling, the cartridge was loaded with 50 µL of coating solution and then blown out with UHP helium. The cartridge was then transferred to an active sampling line, which included an ozone scrubber made from a 0.6-cm section of 1/ -in.-o.d. Teflon tubing packed with crystalline KI and a 2-µm 4 stainless steel screen at the inlet of the sampling line to prevent course particulates from entering the lines. The scrubber is meant to remove ozone because of known positive and negative artifacts.17,18 Air samples were collected at a constant flow rate of 50 mL/min for times of 40-60 min, using a sampling pump and calibrated mass flow controller (Tylan). After sample collection, the sampling cartridge was filled with distilled water and inserted into the sample loop of the injector (Rheodyne 8125). The products of the derivatization reaction, organic-DNPH hydrazones, were then quantitatively swept onto the column for separation and detection. Calibrations were produced by injection of standard solutions of carbonyl hydrazones using a calibrated 5-µL injection loop in place of the sampling cartridge. Software was written using Visual Basic to collect spectral data from the spectrometer and to plot TAS chromatograms (see Results and Discussion). Spectral data were collected at a frequency of 1 Hz. The intensity of each 10 adjacent pixels were averaged and stored, giving an effective pixel group resolution of ∼3.8 nm. Absorbance was calculated for each group of 10 pixels, j, as Aj ) -log(Ij/Io,j), where the matrix of Io,j values was determined by averaging 10 s of data prior to injection. The total absorbance signal was calculated by averaging the absorbances calculated for all pixel groups over the spectral range, 292-473 nm. The TAS was plotted as a function of time, t, and was used for all calibrations. The TAS matrix, Aj,t, is stored to disk such that spectral information can be recovered from chromatographic peaks for qualitative identification. RESULTS AND DISCUSSION Signal-to-Noise Considerations and the Total Absorbance Signal. Using the optical setup shown in Figure 1, individual pixels of the CCD became saturated with an exposure time just greater than 1 s. While a 1-s response time would be sufficiently low for detecting HPLC peaks, the signal-to-noise ratio would not be adequate for good absorbance detection. The manufacturer of the spectrometer quotes a signal-to-noise per element (S/Ni) of 250 when light levels are close to saturation. This S/Ni level is ∼43% of the S/Ni level based upon photon shot noise considerations alone, indicating that shot noise will be a contributing factor to the ultimate detection limit under these light-starved conditions. Practical conditions suggest that the maximum light levels should be maintained at 50-80% of the saturation level. Under these conditions, the S/Ni (1σ) would be in the range of 177-224, assuming a square root relationship between light intensity and S/Ni ratio, and absorbance noise levels would be ∼1.9-2.5 mAU for an individual element (see eq 3). Several options exist to decrease the noise levels. The first would be to increase light levels by using a more powerful light source with a subsequent decrease in exposure time and increased
temporal averaging of the signal. The second option would be to increase light throughput by increasing the aperture of the flow cell. Unfortunately, this would increase the detection volume which is counter to the purpose of the microflow cell. A third option is to keep light levels constant and to average the response of individual diodes in time. The reduction in noise would be proportional to the square root of the number of time periods that are averaged but at the expense of increased response time of the detector. We did not pursue this option, considering our expectation of sharp chromatographic peaks and the need for e1-s response time. A fourth option is to increase the spectral bandwidth of the detector. Bandwidths are typically increased by opening the slits on the monochromator. While the slits used in the monochromator in this study are fixed, the bandwidth can also be increased by averaging the signal from adjacent pixels, an approach commonly used for diode array detectors.8,15 How large the spectral bandwidth can be increased is usually limited by linearity considerations. Beer’s law states that absorbance is proportional to analyte concentration for monochromatic radiation, where monochromaticity implies that the measurement bandwidth is much smaller than the absorption feature of interest. If the spectral bandwidth is increased too much, the absorbance calibration curve shows negative deviations at large analyte concentrations due to changing molar absorptivity of the analyte across the measurement bandwidth in which the logarithm is calculated. Most commercial diode array detectors used for HPLC have spectral bandwidths on the order of 1-16 nm.8 These bandwidths are usually sufficiently small to maintain detector linearity for the broad spectral absorption bands of most organic molecules in liquids. Other than the linearity question, the other reason for using small bandwidths is the perception that greatest sensitivity will be achieved if one chooses the wavelength of maximum molar absorptivity. While this may be so, under shot noise limited conditions, the S/N ratio can be shown to maximize for very large bandwidths that cover a significant fraction of the absorption feature. The issue of nonlinearity can be overcome by calculating the absorbance for small measurement bandwidths, and then calculating the average absorbance, A h , through summation over a much larger spectral bandwidth:
A h)
1
Io,i
n
∑ log I n i)1
i
)
bC n
n
∑ ) i
i)1
maxbC n
n
∑ i)1
i
(2)
max
where the summation is over n CCD pixel elements, Ii is the measured intensity for the ith pixel, Io,i is the reference intensity, b is the path length, C is the analyte concentration, and max is the maximum molar absorptivity. In this paper, A h is referred to as the total absorbance signal. The uncertainty in the absorbance signal for an individual pixel is given by
ai )
1 1 σ ) 2.303 I 2.303S/Ni
(3)
where S/Ni is the signal-to-noise ratio for a pixel. For shot noise limited conditions, S/Ni is simply equal to the square root of the number of photons impinging on the pixel in the measurement period. Note that, in eq 3, we assume that the uncertainty in Io is Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
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Figure 2. Theoretical enhancement of the signal-to-noise ratio (see eqs 5 and 6) with increasing bandwidth for a Gaussian absorption band with standard deviation of σabs nanometers.
Figure 3. Chromatogram of 2-L air sample (40 min) collected in Simcoe, Ontario; 4:00-5:00 p.m., July 19, 2000. Hydrazone peaks: a, nitrite; b, formaldehyde; c, unknown; d, acetaldehyde; e, blank peak; f, acetone; g, glyoxal.
much less than the uncertainty in I. Experimentally, this is achieved by taking many measurements of Io at the start of a chromatogram and using average values of Io,i in eq 2. The noise in the total absorbance signal, A h ( a, is then given by
of an absorbance measurement with a single pixel and increases to a maximum as the measurement bandwidth is increased. The maximum value of f(n,), and thus the bandwidth necessary to give maximum S/NTAS, occurs when the bandwidth is equal to 2.8σabs. This bandwidth is equal to the width of the Gaussian when the value of i has fallen to 1/e (0.37) times max. In this particular case, the signal-to-noise ratio of the TAS increases by more than 1 order of magnitude when the bandwidth of the absorbance measurement is increased from 0.38 to 85 nm. Furthermore, the value of S/NTAS drops off very slowly as the measurement bandwidth is increased further, allowing one to use very large bandwidths to achieve universal absorbance detection without significant reduction in signal-to-noise ratio. With other simulations it was found that the normalized position of maximum f(n,), 2.8σabs, is independent of the pixel resolution, wi, or the width of the absorption feature, σabs, provided that wi , σabs. The optimum bandwidth does depend on the shape of the absorption feature though. Other simulations (not included) showed that the optimum bandwidth is equal to the width of the absorption feature when i has fallen to 1/3max for a triangular-shaped absorption band and 1/2max for a square wave-shaped absorption band.
a)
1 2.303xnS/Ni
(4)
From eq 2, it is apparent that the total absorbance signal is maximum for large path lengths and small values of n centered around the wavelength of maximum molar absorptivity. By contrast, the standard error in the total absorbance signal decreases continuously as one increases the number of pixels over which the signal is measured. More important than the total absorbance signal itself, or the noise in the TAS, is the signal-tonoise ratio for the TAS.
S/NTAS ) 2.303maxbCS/Ni
1
xn
n
∑ i)1
i
(5)
max
One may predict that there will be an optimum value of n or, equivalently, an optimum bandwidth that will maximize the signalto-noise ratio of the TAS. To explore this, the function
f(n,) )
1
n
∑
xn i)1
i
(6)
max
is plotted in Figure 2 for the case of a Gaussian absorption band with standard deviation of σabs nanometers. The function is plotted progressively as the bandwidth of the measurement increases from one pixel element to larger values where the measurement bandwidth is always centered on the Gaussian absorption band. The x-axis has been normalized to the value of σabs. For the particular simulation shown in Figure 2, the parameters used were pixel resolution of 0.38 nm and σabs ) 30 nm, which match our experimental conditions for the spectrometer in use and typical values of σabs for mono-DNPH hydrazones (see next section). The absolute value of the function, f(n,), is equal to 1.0 for the case 1390 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
EXPERIMENTAL RESULTS The absorbance noise limits of the detection system were determined by measuring the standard deviation in the chromatographic baseline when clean solvent was running through the system at 50 µL/min. The standard deviation (1σ) is typically 1.2 × 10-4 AU. As an example, the TAS noise in a clean baseline section of the chromatogram shown in Figure 3 was 1.16 × 10-4 AU. The theoretical absorbance noise can be calculated based upon the values of S/Ni, n, and the average light intensity over the spectral region of interest. For example, the light intensity is typically 50-80% of full scale in the region of 292-476 nm. The number of pixels in the spectral range is 510. The value of S/Ni is 250 for full-scale exposure. Using eq 4, the expected noise is calculated to be 1.18 × 10-4 AU, in good agreement with the measured value. Typical noise levels in diode array detectors used in commercial analytical HPLC’s are in the range of 1 × 10-5-1 × 10-4 AU. This value is dependent on the light source intensity, its stability, and the wavelength of interest. The path length of this cell is significantly longer than other commercial flow cells of similar volume. This longer path length
Figure 4. TAS chromatographic peaks for hydrazones of acetone and glyoxal as a function of spectral bandwidth centered at λ ) 368 nm.
results in better absorbance detection sensitivity. The detection volume of the cell, 30 nL, is suitable for use with micro-HPLC columns of inner diameters greater than or equal to 300 µm. Despite this, we currently use 1.0-mm columns due to a more favorable link of the sampling system to the analytical system. The dead volume using a 20-cm section of 0.005-in.-i.d. PEEK connecting tubing is ∼240 nL. This is acceptable for use with 1.0-mm columns since the sum of the detection volume and the connecting volume is only 270 nL, much lower than the limit of 400 nL outlined in the introduction. For use of this cell with capillary columns, however, the connecting volume should be reduced to eliminate the potential for extra column band broadening. This could be accomplished by using narrow-inner diameter fused-silica capillary in the flow cell design, rather than PEEK tubing for the connection to the column. As an example of the performance of the flow cell and detection system, Figure 3 shows a chromatogram of an air sample collected in a recent field study in a remote area of southern Ontario. The major identified components in the air sample include formaldehyde (0.96 ppbv), acetaldehyde (0.59 ppb), acetone (1.4 ppb), and glyoxal (0.60 ppb). The peaks have been identified both by retention time and from the corresponding absorbance spectra. For illustration purposes, the peaks corresponding to representative hydrazones, acetone and glyoxal, are given special attention in the following analysis. The background-subtracted absorbance spectra (10 s average) of acetone and glyoxal obtained from the chromatogram are shown in Figure 5. The spectra are in good agreement with spectra found in the literature.19 Acetone-DNPH (17) Smith, D. F.; Kleindienst, Tadeusz E.; Hudgens, E. J. Chromatogr. 1989, 483, 431-6. (18) Arnts, R. R.; Tejada, S. B. Environ. Sci. Technol. 1989, 23, 1428-30. (19) Druzik, C. M.; Grosjean, D.; Van Neste, A.; Parmar, S. S. Int. J. Environ. Anal. Chem. 1990, 38, 495-512.
Figure 5. Background-subtracted absorbance spectra (10 s average) for peak f (acetone-DNPH) and peak g (glyoxal-bis-DNPH).
Figure 6. Chromatographic peak height (net) signal-to-noise ratios for peak f and peak g as a function of spectral bandwidth centered at λ ) 368 nm.
has a wide absorption band (fwhm ) 69 nm) with peak absorbance at 368 nm while glyoxal-bis-DNPH has an unsymmetrical absorption band (fwhm ) 115 nm) with peak absorbance at 435 nm. To compare to the theory outlined previously, the TAS signal was calculated for varying spectral bandwidths centered at wavelength of maximum molar absorptivity for acetone, 368 nm. The effect of the bandwidth on the two chromatographic peaks is shown in Figure 4 for bandwidths from 3.8 (1 group of 10 pixels) to 157 nm (43 groups of 10 pixels). As the bandwidth increases, the chromatographic peaks become smoother, simultaneous with a slight reduction in the peak height. To assess the impact of the bandwidth on the signal-to-noise ratio, peak heights and peak areas were calculated for both of the chromatographic peaks as a function of bandwidth. The noise was measured in a clean section of each of the chromatograms. The corresponding S/NTAS ratios for peak height and peak area are shown in Figures 6 and 7, respectively. The S/NTAS for acetone maximizes at a bandwidth of ∼55-84 nm. The S/NTAS for glyoxal appears to be approaching a maximum at the largest bandwidth of 157 nm. The S/NTAS curve Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
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Figure 7. Chromatographic peak area signal-to-noise ratios for peak f and peak g as a function of spectral bandwidth centered at λ ) 368 nm.
in Figure 6 for acetone, whose absorbance spectra most closely resembles a Gaussian, matches that seen in Figure 2 in several ways. Initially as the bandwidth is increased, there is a rapid increase in the S/NTAS ratio. Since the molar absorptivity is almost constant across the spectrum in this region, the increase in S/NTAS is proportional to the square root of the number of pixels or, equivalently to the square root of the bandwidth. The function then maximizes at ∼1.9-2.9σabs compared to a value of 2.8 σabs predicted by theory for a Gaussian absorption band (Figure 2). There is a gradual decrease in the S/NTAS value as the bandwidth is increased further beyond the maximum. The expected S/NTAS enhancement for the grouping of 10 pixels is x10. Taking this into account, the maximum S/NTAS enhancement is 8.5 compared to the S/NTAS for an individual pixel. This compares reasonably well to the maximum value of 11.0 seen in Figure 2. The signalto-noise ratio enhancements for glyoxal are larger than those for acetone and exhibit a maximum at a much larger bandwidth due to the larger spectral width of the absorption band. The S/N ratios are larger for peak areas compared to peak heights (compare Figure 7 to Figure 6). The larger values of S/N for peak areas arise from the time-averaging benefit when one integrates the chromatographic peak. Despite the larger value of S/N for peak areas, the signal-to-noise ratio enhancements seen as the spectral bandwidth is increased are almost identical for peak areas and peak heights. The operational TAS bandwidth used in this study (181 nm) has been chosen for the specific application of separation and detection of organic carbonyl hydrazones, including the hydrazones of saturated aldehydes, saturated ketones, aromatic aldehydes, dicarbonyls, and other multifunctional species. Although this bandwidth is somewhat larger than the theoretical observed maximum for monocarbonyl hydrazones, it achieves a more universal hydrazone absorbance detection. In addition, the calibration response of the large-bandwidth TAS is similar for many bishydrazones and is roughly twice that for many monohydrazones,
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allowing a reasonably accurate calculation of concentrations even for unknown peaks, provided that one can differentiate between mono- and bis-DNPH products. Total absorbance signal (181-nm bandwidth) calibration curves for the hydrazones were found to be linear over a large concentration range from the detection limit to at least 3 orders of magnitude higher. At the upper end, negative deviation is observed when absorbance approaches a value of 1, likely due to dark noise or stray light in the spectrometer. At the lower end, the TAS detection limit of the cell (3σ) is 3.6 × 10-4 AU. For the DNPH hydrazones, this corresponds to a detection limit (3σ) of ∼(420-670) × 10-15 mol using the known amount of material injected and the S/N ratios derived from the chromatogram in Figure 3. For a 2-L air sample collected on the cartridge, this instrumental detection limit corresponds to ∼5-8 ppt of organic carbonyl in air. CONCLUSIONS The UV/vis flow cell described in this paper is simple but very sensitive. The small aperture of the flow cell (2.5 ×10-3 mm2) limits the light throughput such that shot noise is a limiting source of noise for the absorbance detection limit of an individual pixel. The absorbance S/N ratio has been increased in this case through the use of large-bandwidth total absorbance signals that are much larger than bandwidths commonly used in HPLC detection. The TAS detection scheme can be used in any optical multichannel device, including diode array detectors, and would be especially useful for other small-aperture flow cells used in microcapillary separation techniques where light throughput is a limiting factor for the detection limit. The optimum TAS bandwidth necessary to optimize the S/N ratio is ∼1.0-1.5 times the expected fwhm of the absorption feature of the analyte. Beyond this optimum bandwidth, the S/N ratio decreases slowly. Besides the improvement in S/N ratio, the benefits of using larger bandwidth signals are as follows: (i) more universal absorbance detection in the chromatogram and (ii) similarity in response factors for absorption chromophores despite shifting of peak molar absorptivity with different derivatives on the chromophore. The disadvantage of using a large-bandwidth TAS absorbance signal is the loss of selectivity, but since the spectral information necessary to compute the TAS is stored to disk, the option for selectivity is never lost. Software modifications could easily be made to allow a user to choose a wavelength and spectral bandwidth post facto to the running of the sample, to optimize S/N ratio and selectivity for the displayed chromatogram and for use in calibrations. ACKNOWLEDGMENT Funding was provided by the National Science and Engineering Research Council of Canada. Received for review August 9, 2000. Accepted January 7, 2001. AC000943E
