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Anal. Chem. 1088, 60, 2422-2427
Sensitivity Improvement in Infrared Detection for Supercritical Fluid Chromatography Richard C. Wieboldt* and Gregory E. Adams Spectroscopy Research Center, Nicolet Instrument Corporation, 5225 Verona Road, Madison, Wisconsin 5371 1
Douglas W. Later Lee Scientific, Inc., 4426 South Century Drive, Salt Lake City, Utah 84123
Dramatlc Improvements in the sensltlvity of Fourier transform infrared detectors for supercritical fluid chromatography are explained In terms of optical and chromatographic parameters. By use of methyl palmitate as a model compound, minimum ldentlkation HmHs of 10 ng of component delivered to the column using carbon dioxide mobile phase are reported. Detectlon limits such as these are achieved by balancing optical throughput of a 600 q internal diameter flow ceU against the effect of moMle phase absorption with a 5inm path length. Results from a study of the detector cell temperature indicate an improvement In sensJtivity can also be attributed to concentration of the chromatographic peak when using cooler detector cell temperatures.
Supercritical fluid chromatography (SFC) is attracting a great deal of interest as a powerful new technique in the field of chromatographic analysis. One of the primary areas of current research in SFC is the development of full range spectral detectors such as Fourier transform infrared (FT-IR) spectroscopy. This is an important development because FT-IR detectors with SFC can provide full infrared spectral information for identification of chromatographic peaks. Recently, there has been a dramatic improvement in the performance of FT-IR flow cell detectors with SFC. While this improvement has generated considerable interest, there has been little explanation of how these improved detection limits were obtained or the factors affecting detection limits. This paper addresses these concerns and reports results from experiments that study the optical and chromatographic parameters involved in the SFC/FT-IR experiment. By use of model compounds, the identification capability and chromatographic performance of the flow cell type of SFC/FT-IR interface are defined. A recent review by Jinno ( 1 ) describes past efforts in the area of FT-IR detection in SFC. There are two basic approaches to the problem. One involves deposition of the effluent on a substrate for subsequent FT-IR analysis (2-7); the other employs the flow-through cell approach (8-12). Each technique has its strengths and both should be evaluated for their suitability in solving a given analytical problem. This paper addresses only the flow cell type of interface. One of the primary advantages of flow cell detection is simplicity of use. From a conceptual viewpoint, the cell acts as any other chromatographic detector. It attaches to the end of the chromatographic column and provides an analytical signal in real time. This signal is directly proportional to the amount of sample in a chromatographic peak flowing through the cell at the given time. Detection is nondestructive and allows the use of additional detectors connected in series if the cell is designed such that the volume does not drastically compromise chromatographic resolution. The primary disadvantage of the flow cell approach is distinguishing mobile phase absorbance from sample absor0003-2700/88/0360-2422$01 S O / O
bance. This has been the nemesis of FT-IR detection with HPLC where the mobile phase absorbance is so great that flow cell detection is virtually impractical. As a mobile phase, carbon dioxide is very compatible with infrared detection because its absorption bands lie in regions of the IR spectrum that are rarely critical for identification of unknowns. Other mobile phases, such as xenon proposed by Novotny et al. (12), are ideal for infrared detection because of their lack of absorption bands. However, carbon dioxide is currently the most widely used mobile phase for supercritical fluid chromatography. A suitable FT-IR detection cell should be compatible with COz under the density conditions typically used for SFC.
EXPERIMENTAL SECTION Flow Cell. The flow-throughanalysis cell body is constructed from a single piece of stainless steel with collars attached for holding the window and seal assembly (Figure 1). The central bore has a 600-pm internal diameter and a 5-mm path length. This gives a cell volume of 1.4 pL. Two channels at either end of the central bore connect the bore to transfer line fittings machined into the sides of the cell body. Fittings are designed so that the transfer line seal is made inside the cell body close to the central bore. The fused silica transfer lines, each being 0.5 m x 50 pm i.d., are sealed by captive graphite ferrules in the nut. During assembly, the transfer line is inserted through the connecting channel such that the end is flush with the inner surface of the central bore. The inside diameter of the connecting channel is closely matched to the fused silica outside diameter. This effectively eliminates the connection channel dead volume that would be present if the transfer line terminated at the fitting. Infrared transparent windows are attached to the cell by using fluoroelasticgaskets between the window and cell body. A hollow compression screw applies force to the window and gasket assembly forming a positive seal capable of withstanding pressures exerted by 425 atm of carbon dioxide. The cell is held in the optical beam with an insulated, temperature-controlled mount. For the experiments described here, the transfer lines were not controlled but left at ambient temperature. Optical components are selected to focus a collimated beam from the FT-IR optical bench on the flow cell aperture. Exiting radiation is collected by similar optics, which match the beam to the detector element of the mercury-cadmium-telluride (MCT) detector. FT-IRSpectrometer. A Nicolet 2OSXC FT-IR spectrometer (Nicolet Instrument Corp., Madison, WI) was used in these experiments. Real time FT-IR spectra were collected at 8-cm-' resolution by using the Nicolet Specific Infrared Detector (SID) software package. Eight scans were coadded per file and stored on magnetic disk. Time resolution between stored files was 1.1 s. For each chromatographic peak, stored spectral files were coadded as necessary to provide the optimum signal-to-noise ratio in the final absorption spectrum. SFC Chromatograph. A Lee Scientific Model 501 supercritical fluid chromatograph (Lee Scientific,Salt Lake City, UT) was used for pressure testing the cell and for chromatographic separations. For the dimethylpolysiloxaneexperiments, a 200-nL injection of neat dimethylpolysiloxanefluid (molecular weight 3600) was split 25:l and separated on a 10 m X 50 pm id., 0.25-pm film SB-Methyl-100 column (Lee Scientific) using pure SFC grade 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
2423
Peaden and Lee (13)treated acceptable cell volumes more rigorously by using the following expression to estimate allowable detector volume in capillary SFC:
Vi = 0 . 8 6 6 ~ d , 2 ( L H ) ~ / ~ [-l / ARJ2 (l - 1I1l2(1+ k')
(1)
In the above equation, Vi is the volume of the detector, AR8 is the fractional resolution loss, L is the column length, H is the plate height, k'is the capacity factor, and d, is the internal column diameter. As a specific example of its implementation for capillary SFC, the calculated allowable detector volume resulting in a 1% resolution loss for a 20 m x 100 pm i.d. column, with a plate height of 0.6dc and a k'value of 1, is 0.27 pL. This is a factor of 5 smaller than the current flow cell design; hence, greater than 1% loss in resolution is expected. These small cell volumes can be attained, but only a t the expense of detector path length and sensitivity. Fields and Lee (14) further extended eq 1 to include the effects of peak compression by including a fractional density factor, p, for the column vs detector
I
Flguro 1. SFC/FT-IR flow through detector cell: (1) hollow compression screw: (2) collar attachment bo& (3) collar; (4) window holder; (5) window: (6) gasket; (7) cell body; (8) ferrule; (9) transfer line fitting; (10) thermocouple well; (1 1) cartridge heater well.
carbon dioxide (Scott Specialty Gases, Plumstead, PA). The oven temperature was programmed from an initial temperature of 100 to 120 O C at 1.5 OC/min after a 22.67-min initial hold. This temperature was held for 8.00 min and then ramped at 1.5 OC/min to a final temperature of 160 O C . The COPwas density programmed by using the multistep method listed in Figure 3. The fluid densities listed in Figure 3 assume a constant oven temperature of 100 O C . Because the oven temperature was programmed to 160 O C , the actual fluid densities in the capillary column were less than these values. For the methyl palmitate experiments,standard solutions of methyl palmitate in dichloromethane were prepared at 0.20,0.10, and 0.05 mg/mL concentrations. Each sample was injected using direct injection of a 200-nL sample loop. A 10 m X 100 pm i.d., 0.50-pm film SB-Methyl-100 column (Lee Scientific) was used for each run. Pure COzat 100 "C was density programmed from 0.20 to 0.36 g/mL at 0.01 (g/mL)/min after a 10-mininitial hold. The methyl palmitate eluted at 23 min corresponding to a density of 0.33 g/mL at 100 O C . Because the flow cell was thermostated at 35 "C, the actual fluid density in the flow cell was 0.82 g/mL. The 1%by weight l-hexanol in supercritical grade COz (Scott Speciality Gases) measurements were made with the flow cell at 35 "C. The SFC chromatograph was used to deliver the modified mobile phase to the flow cell at 100 and 400 atm.
RESULTS AND DISCUSSION Flow Cell Dimensions. The design of any flow-through detector cell is a compromise between the conflicting requirements of an absorbance based detector and a chromatographic detector. From a chromatographic viewpoint, the primary requirement for efficient detection is that detector cell volume be small relative to the volume of the chromatographic peak. Typical peak volumes for a 100 pm i.d. capillary column operated at a linear velocity of 2 cm/s range from approximately 1.5 to 9.4hL, assuming base peak widths of 10-60 s. For the 1.4-pL flow cell design described here, the ratio of peak volume to detector volume is in the range 1.1-6.7. In contrast, typical peak volumes for a 1 mm i.d. packed column under the same conditions range from approximately 150 to 942 pL. Therefore, the peak volume to detector volume ratio is between 107 and 673 or 2 orders of magnitude greater. Capillary columns impose much more stringent detector dead volume requirements and limitations.
Hence, if the column and detector densities are the same, there is little impact on required detector cell volume, V,, to maintain a given resolution. But, if the density in the flow cell is greater than in the column due to cooling, for example, the required detector volume for the same resolution is decreased. Absorbance detection methods such as FT-IRare based on the Beer-Lambert law. The amount of absorbance is proportional to the sample molar extinction coefficient, the cell path length, and the sample concentration. Of these three factors, cell path length is the only parameter that affects FT-IR absorbance. The molar extinction coefficient is a sample-dependent constant, and concentration is a function of the chromatographic separation. Although a larger path length increases the sample absorbance, it also contributes to a larger detector volume. If cell path length and volume were the only factors involved in cell design, the optimum flow-through absorption cell would have an infinitely long path length and an infiitely small cross section for zero dead volume. In reality, restrictions imposed by the optics of the detector system dictate the cell diameter and hence the detector cell volume. For a cell having a fixed volume, decreasing the cross sectional diameter while at the same time increasing the path length to keep the cell volume constant will increase absorbance. However, the amount of energy transmitted by the cell (throughput) decreases. This decreased throughput causes the noise level in the recorded spectrum to increase. Since detection is based on the signal-to-noise ratio of an absorption band in a spectral base line, an optimum point is reached between increased absorbance (signal), through the use of smaller diameter, longer path length cells, and optical throughput (noise). The volume of the flow cell can be reduced if the internal diameter of the flow cell is decreased as far as possible while still maintaining adequate energy throughput. From the Beer-Lambert law, it is apparent that the internal diameter of the cell does not affect sample absorbance. The only limitation to internal diameter is optical throughput. By use of state of the art optical components, the minimum practical diameter was found to be 600 pm. Diameters smaller than this reduce throughput to the point where the spectral noise level becomes unacceptable for typical applications. With SFC/FT-IR, one must also consider the effect of the mobile phase absorption. The optimum path length of the flow cell is strongly dependant on the mobile phase. For supercritical carbon dioxide, the asymmetric 0-C-0 stretch absorbs all energy between 2551 and 2137 cm-' as do the
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER
1, 1988
combination bands between 3822 and 3504 cm-'. The absorption bands between 1475 and 1225 cm-' are caused by Fermi resonance between the first overtone of the 0-C-0 bending fundamental at 667.3 cm-' and the 0-C-0 symmetric stretching fundamental at 1334 cm-'. This gives rise to a band at 1382 cm-' and a doublet at 1287 and 1277 cm-'. These absorption bands are often referred to as the Fermi bands. The intensities of the carbon dioxide absorption bands increase with density of the supercritical fluid. However, it is only the Fermi bands that are crucial for flow cell FT-IR detection. The other bands are totally absorbing and leave no useful energy for sample characterization. Morin et al. (15) showed that the Fermi band intensities increase with density according to the equation
A = a exp(Pp) (3) where p is the density of carbon dioxide and a and p are constants that differ slightly for each Fermi band. In the density range from 0.8 to 1.0 g/mL, the infrared absorbance of the Fermi bands increases by a factor of 2. Carbon dioxide absorptivity measurements obtained in our laboratory with cell path lengths of 7.5 and 5 mm gave results that were consistent with the data measured with a 10 mm path length cell reported by Morin. In order to use carbon dioxide mobile phase at high densities (1.0 g/mL), the cell path length must be short enough so that the Fermi resonance bands do not absorb all the infrared energy. Absorption bands that are stronger than 1absorbance unit (10% transmission) yield poor results during spectral subtraction when data are collected by using the SFC/FT-IR experimental conditions described in the previous section. There is simply not enough energy throughput to provide an adequately low noise level in the subtracted result. With this criterion of 1 absorbance unit as a maximum allowable absorbance for the Fermi resonance bands at a maximum working density of 0.9982 g/mL (31.5 OC at 425 atm), the allowable cell path length is 5 mm. Path lengths longer than this will produce too much absorbance by the Fermi bands and all useable energy in the Fermi resonance region of the spectrum is lost. This path-length limitation is a function of the mobile phase absorbance. For supercritical fluids that are infrared transparent, such as xenon, the path-length restriction imposed by mobile phase absorbance does not apply. However in these cases, the path length is still limited by energy throughput. Increasing the cell path length severely reduces the amount of energy that is transmitted through the cell. This lower throughput, as mentioned earlier, results in a hgher spectral base line noise level. Even though there is a linear gain in sample absorbance by increasing the path length, there is a much faster gain in background noise level because of decreased throughput. The net result is poorer overall signalto-noise ratio. To alleviate this problem, the cell diameter can be increased, but this rapidly increases the cell volume making it unsuitable for chromatographic applications. In some SFC applications, modifiers are blended with the pure carbon dioxide mobile phase to alter the chromatographic separation. These are usually polar organic compounds, such as methanol, at concentrations typically in the 1-10% by weight range. Although the mechanism by which a modifier affects chromatographic separation is not well understood, it is useful to examine the effect of modifiers on flow cell infrared detection. Any organic modifier has infrared absorption bands that can become totaUy absorbing at high concentrations. For this reason, 1-hexanol was selected as modifier because it has a high solvatochromic polarity relative to the more commonly used modifiers such as methanol (16). In theory, this means that a 1% concentration of hexanol, for example, can achieve
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Infrared absorption of 1% l-hexand modifier in supercritical carbon dioxide: (a) 100% T spectral base line from a 400 atm single-beam spectrum ratioed to a 100 atm single-beam spectrum; (b) single-beam absorption spectrum measured at 100 atm and 35 OC. the same modifying effect as a 6% concentration of methanol. Figure 2b shows the infrared absorption of a 1% hexanol modified carbon dioxide mobile phase measured at 100 atm and 35 "C by using the flow cell described above. It is apparent from the negative-goingabsorption bands in this single-beam spectrum that large regions of the mid-IR are blocked by the mobile phase. Figure 2a is the spectral base line obtained by ratioing a single-beam spectrum measured at 400 atm to a single-beam spectrum measured at 100 atm. The blank regions in this 100% T line are those portions of the spectrum where all useful energy is absorbed by the mobile phase. The solid lines between the wavenumber annotations represent the "windows" that can be used for infrared detection. Clearly even low levels of modifier can present serious problems with mobile phase absorption in selected regions of the infrared spectrum. Applications using modified C02will be limited to those in which the infrared absorption bands of interest lie in one or more of the mobile phase window regions. FT-IR Detection with Packed Columns. Unlike the case for gas chromatography (GC)/FT-IR, the optimum cell dimensions for SFC/FT-IR detection are the same for both packed and capillary column applications. This arises primarily from the cell path length criteria. Although packed column SFC peak volumes are approximately 2 orders of magnitude greater than for capillary columns, the path length and volume of the cell cannot be increased, as they can be in GC/FT-IR, to obtain improved sensitivities. This is because chromatographic dead volume considerationsare overshadowed by the necessity to maintain short flow cell path lengths due to absorbance of the mobile phase. In GC/FT-IR, there is no mobile phase restriction on cell path length since commonly used carrier gases (e.g. hydrogen and helium) do not absorb in the mid-IR region. Therefore, the cell can be designed to have as long a path length as possible without the volume exceeding the typical chromatographic peak volume. In these situations, the larger peak volumes associated with packed column GC allow for the use of a larger volume cell. In principle then, the cell path length could be made even longer because the diameter can be increased to let more energy through the longer path length cell. It is important to note that merely enlarging the cell diameter to match peak and cell volume does not improve detector performance;the path length has not changed. The exception is in energy-limitedsituations. In these cases, a larger diameter will increase throughput, which reduces the background noise level and thereby improves the overall signal-to-noise ratio. However, an optical detector system properly designed to Flgwe 2.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 2425
obtain adequate signal-to-noise performance with a small diameter cell is not an energy-limited situation. To summarize then, the only advantage of increasing cell volume to match the volume of packed column peaks is to obtain greater absorption path length. For supercritical carbon dioxide, the maximum practical path length is fixed. Because of this, a flow cell optimized for FT-IFt detection with capillary carbon dioxide SFC is also optimum for packed column applications. In terms of chromatographic performance, the same cell should actually perform better for packed column applications because its effect on chromatographic resolution is less. Temperature Control. The flow cell and transfer lines are under separate temperature control. The transfer lines can be maintained at any constant temperature. The flow cell is limited to a maximum temperature of 50 "C. Early work with this interface used no temperature control; i.e. the cell and transfer lines were a t ambient temperatures ranging anywhere from 22 to 31 "C depending on laboratory conditions. Since these temperatures are below the critical point for COz, FT-IR detection was probably at subcritical conditions. As the fluid is pressurized, it goes through an opalescent or milky transition where infrared transmission is severely curtailed. The ambient cell temperature conditions were above this opalescence point. Studies with the cell and transfer line maintained at supercritical temperature of 35 "C showed essentially no difference in chromatographic resolution compared with studies at 22 "C. This can be explained by the effect of temperature on the carrier fluid. At low temperature, the density of the fluid increases, which increases its solvating power. Any solutes dissolved in the mobile phase should therefore remain in solution even though the temperature drops. One would also expect to maintain better chromatographic peak shape when the interface is at low temperature. The diffusion coefficient of a given analyte in the mobile phase decreases with decreasing temperature at constant density. There is also little interaction between the mobile phase and the capillary and flow cell surfaces because the transfer lines and cell are uncoated. As a result, there is less longitudinal band broadening in the mobile phase, which is advantageous. To verify this hypothesis, a sample of dimethylpolysiloxane having an average molecular weight of 3600 was used as a sample probe. Figure 3a is the flame ionization detector (FID) chromatogram of the sample separated with a combined temperature/density program when the flow cell is not connected to the chromatograph. Figure 3b is the same separation with the flow cell connected in series with the FID. The flow cell and transfer lines were left at ambient temperature of 22 "C. Figure 3c is the same analysis performed with the flow cell and oven temperature at 100 "C. This run was aborted after the hot cell developed a leak at higher pressure. Figure 3a shows good efficiency throughout the separation. However, Figure 3b shows two interesting features: (1) there is an obvious loss in resolution as displayed by the broader peaks; and (2) the resolution loss is more severe at lower densities than higher density. For example, compare peak widths of oligomers n = 10 and n = 60 from both chromatograms; the n = 60 peak widths are comparable but the n = 10 peak widths are very different. Both observations are related to the volume of the cell. The cell volume constraints are more stringent at lower densities than at higher densities. For example, to maintain a loss of no more than 1% in resolution for a 10 m X 50 wm i.d. capillary column requires a flow cell volume of no more than 54 nL at a density of 0.2 g/mL. But, for the same resolution loss, a cell volume of 117 nL can be tolerated with the same column system at a density of 0.8 g/mL. This is more than a 2-fold increase in tolerable
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Figure 3. (a) FID chromatogram of dimethylpolysiloxane SFC separation without FT-IR flow cell connected (densities calculated at 100 "C): initial density, 0.19 g/mL initial hold time, 12.00 min; density ramp 1, 0.15 (g/mL)/min to 0.35 g/mL; density ramp 2. 0.01 (g/mL)/mln to 0.54 g/mL; density ramp 3, 0.007 (g/mL)/min to 0.60 g/mL; density ramp 4, 0.004 (g/mL)/min to 0.65 glmL; density ramp 5, 0.002 (g/ mL)/min to 0.70 g/mL. (b) Same separation with FT-IR flow cell connected,transfer lines and cell at ambient (subcritical)temperature. (c) Same separation with transfer lines and FT-IR cell thermostated at 100 "C.
cell volume based on the operating density of the system. An additional advantage to ambient temperature detection is the "peak compression" phenomenon described by Smith et al. (17). With the temperature reduced from 100 to 25 "C at a constant pressure of 200 atm, the density of COz increases from 0.4915 to 0.9224 g/mL. The chromatographic peak is partitioned between the stationary and mobile phases according to the capacity factor, k' n mol of solute in stationary phase k' = (4) n mol of solute in mobile phase In the flow cell and transfer lines, there is no stationary phase so the capacity factor approaches zero and all the solute is contained in n mol of mobile phase. Because of the higher density just mentioned, these n mol of mobile phase are now contained in a smaller volume. For example, the volumetric flow rate at a density of 0.2 g/mL under SFC conditions (50 "C) is 9.4 wL/min. The same supercritical flow rate is reduced
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
to 2.0 pL/min under liquid conditions (0 "C) at the same pressure. This phenomenon, of course, is again a function of density with a transition under similar conditions from 9.4 to 7.2 pL/min at a density of 0.8 g/mL. This volumetric compression in flow rate also leads to a concomitant analyte peak volume compression. The net result is that the concentration of solute molecules in the chromatographic peak is increased which enhances detection. The effect of density change on chromatographicresolution can be explained by considering the volume of the flow cell in relation to the volume of mobile phase containing the peaks. Assume we have resolution between two chromatographic peaks eluted with 1.4 p L of mobile phase, the same volume as the flow cell. If we also assume complete mixing of the mobile phase on passing through the cell, these two peaks will loose chromatographic resolution and elute together. The same principle applies to chromatographic peaks undergoing "peak Compression" associated with the mobile phase density increase resulting from the cool detector cell. The flow cell volume is a constant, but the peak elution volume is decreased. Therefore, we expect a slight decrease in chromatographic resolution because the cell volume now has a greater relative effect on the smaller volume peaks. The "peak compression" effect has a more pronounced effect on narrow chromatographicpeaks. Once a peak is compressed to a smaller elution volume and then expanded,the whole peak is spread out over the expanded volume. The flow cell is simply introducing an extracolumn mixing effect, which imposes a minimum volume on everything flowing through it. In this case, the minimum volume for a peak after the SFC/FT-IR flow cell is 1.4 pL at high density regardless of the volume of the original peak at a typically lower density. This can be rigorously treated as a form of extracolumn band broadening as described by Snyder and Kirkland (18). This can be verified experimentally by comparing the chromatograms in Figure 3b run at 22 "C and Figure 3c run at 100 "C. Note that the early eluting peaks retain their resolution better when not compressed; that is, when the cell is heated at 100 "C compared with the ambient cell. The peak broadening is the ratio of the compressed to noncompressed densities, which, early in the run at low densities, is a t its largest value. This is one reason why the early peaks suffer more resolution loss than the latter peaks. For example, suppose a peak elutes a 1.9 pL of mobile phase-a typical value. At 100 atm and 100 "C, the peak volume is compressed to 0.42 pL with the cell a t 25 "C. Because this is less than the volume of the cell, the peak is effectively broadened to the cell volume by mixing. The assumed 1.4-pL peak volume is a minimum because as the front of the peak (i.e., the first few tenths of a p L of the peak) starts to elute into the cell volume, it is immediately diluted with the existing 1.4 pL which is exiting the cell at -10 pL/min. Thus, by the time the end of the peak elutes, there is probably more peak band broadening than 1.4 pL. When expanded back to 100 "C, this volume further increases. As a result, the effective cell volume is actually 6.2 pL. For the same peak eluting at higher density such as 300 atm and 100 "C, the compressed peak volume is 1.3 pL. Because this is essentially the volume of the cell, there is no peak broadening effect resulting from compression. The only broadening effects are from the actual cell volume. It is apparent that a cool detector cell is advantageous for absorbance-based detectors because of the concentrating effect. In terms of chromatographic resolution, a cooled detector cell is not desirable because of its peak broadening effect at low densities. However, these constraints may be balanced against one another in the cell design. It should be pointed out that very small volume absorption cells will benefit from the compression effect because it increases sample concen-
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tration. Since the cell volume is so small this may outweigh the detrimental effects of compression/expansionbroadening. Sensitivity. As with any chromatographic detector, the sensitivity of the flow cell SFC/FT-IR interface is very much a function of the chromatographic peak shape. A narrow chromatographic peak where all the sample is concentrated in a small volume of mobile phase gives a larger absorbance than a broad peak where the same quantity of analyte is diluted over a large volume of mobile phase. To avoid this dependence on peak shape, chromatographic detectors are usually specified as having detection limits that are defined as a signal that is 3 times the root-mean-square noise level. A detection limit specification such as this is meaningless for FT-IR detection. The reason for using FT-IR as a detector is to obtain spectral information about the analyte, not simply detection. Hence the concept of minimum identification limits (MIL) has been proposed for spectral detectors. Shafer and co-workers (4) have defied MIL as the quantity of compound required for identification by spectral interpretation or computer search. Although the criteria for measuring MIL are subjective, the concept is a good one and more useful than a detection limit. To establish an MIL for the flow cell interface described here, we used methyl palmitate as a model compound. The sample was delivered to the capillary column by using direct injection. Previous work with split injections gave varying split ratios depending on the type of solvents used and initial chromatographic conditions. Although reproducibility was good, there was no measure of exactly how much material was delivered to the column. By use of direct injection, the amount of material cannot exceed the amounts reported for a full sample loop. The actual amount delivered to the column may be less than that reported if the full loop volume was not be delivered in the 0.6-s injection time. SFC/FT-IR absorption spectra of methyl palmitate at lo-, 20-, and 40-ng levels of compound delivered to column are shown in Figure 4. These spectral are plotted on the same scale for comparison. For each spectrum, spectral files collected across the chromatographic peak were coadded as necessary to obtain the best signal-to-noise ratio in the final spectrum. The carbon dioxide mobile phase absorption was
2427
Anal. Chem. 1988. 60. 2427-2435
then removed by using spectral subtraction. The absorbance of the carbonyl peak in the final spectra is perfectly linear. This indicates that the spectral coaddition method can be reliably used for quantitative measurements. The spectra features necessary for identification of methyl palmitate are visible a t the 10-ng level. With this criterion for an identification limit, this is an MIL of 10 ng. Application. The SFC/FT-IR flow cell interface described here has been used in a variety of applications including the analysis of thermally labile carbamate pesticides (19-21) and natural products (20,22). In each application, FT-IR identification of the eluted components shows high sensitivity for these compounds and provides positive identification to distinguish between related species.
Raynor, M. W.; Bartie, K. D.; Davies, I. L.; Williams, A.; Clifford, A. A,; Chalmers, J. M.; Cook, B. W. Anal. Chem. 1988, 60, 427-433. Shafer, K. H.; Griffiths, P. R. Anal. Chem. 1983, 55, 1939-1942. Oiesik, S. V.; French, S. B.; Novotny, M. Chmmatcgraphie 1984, 18,
489-495. Johnson, C. C.; Jordan, J. W.; Taylor, L. T.; Vidrine, D. W. Chromatograph& 1985, 20, 717-723. Hughes, M. E.; Fasching. J. L. J. Chromatogr. Sci. 1985, 2 4 ,
535-540.
Shafer. K. H.; Pentoney, S. L.; Griffiths, P. R.; Fuoco, R. HRC CC, J . High Resolut . Chromatogr . Chromatogr Commun 1988. 9 ,
French, S. B.; Novotny, M. Anal. Chem. 1988, 58, 164-166. Peaden. P. A.; Lee, M. L. J. Chromatogr. 1983, 259, 1-16. Fiekls, S. M.; Lee, M. L. J. Chromtogr. 1985, 349, 305. Morin, P.; Caude, M.; Richard, H.; Rosset, R. Chromatograph& 1988, 2 1 , 523-530. Levy, J. M.; Rltchey, W. M. HRC C C , J . High Resolut. Chromatogr. Chromatogr. Commun 1987, IO, 493-496. Smith, R. D.; Kaiinoski. H. T.; Udseth, H. R.; Wright, B. W. Anal. Chem. 1984, 56, 2476-2480. Snyder, L. R.; Kirkland, J. J. Introduction to Modern UquH Chromatography, 2nd ed.; Wiley: New York, 1979; pp 31-33. Wieboidt. R. C. Ana&& of PestlcHes by Cap//&ry SFC-FTIR; Nicolet FTIR Application Note AN-8705, 1967. Wieboldt, R. C.; Smlth, J. A. I n Supercrltical FluH €xtraction and Chromatography-Techniques and Appiicabns ; Charpentler, B. A., Sevenants. M. R., Eds.; ACS Symposium Series 366 Amerlcan Chem ical Soclefy: Washington, DC, 1988; pp 229-242. Later, D.W.; Bornhop. D. J.; Lee, E. D.; Henion, J. D.; Wieboldt, R. C. LC-GC 1987. 5 . 804-816. Wleboldt, R. C.; Kempfert, K. D.; Later, D. W.; Campbell, E. R., submitted for publication in HRC CC, J. High Resolut. Chromatogr. Chromatogr Common.
Pentoney, S. L.; Shafer, K. H.; Griffiths, P. R. J. Chromatogr. Sci. 1988, 2 4 , 230-235.
RECEIVED for review March 25,1988. Accepted August 2,1988.
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Quantitative Effects of an Absorbing Matrix on Near-Infrared Diffuse Reflectance Spectra Jill M. Olinger and Peter R. Griffiths*
Department of Chemistry, University of California]Riverside, California 92521
The absorptlon propertles of a matrix surrounding an analyte influence the band intenSny In near-lnfrared dmuSe reflectance spectra. I f the matrlx does not absorb radiation at the same wavelength as the analytical band, then use of the KubelkaMunk equation provldes a linear relationship between band lntenslty and concentration over a major portlon of the concentration range for the analyte. If the m a t h surrounding the analyte absorbs radlatlon at the same wavelength as the analytical band, then deviations from llnearlty of plots of the Kubeika-Munk function versus concentration occur. I n thls case, the use of log l/R’values instead of the Kubelka-Munk function has been shown emplrlcally to provide a more linear relatlonshlp between reflectance and concentration. I t has also been shown that the concentration range over which llnearlty holds Is dependent upon partlcle sire and on the strength of the absorptlon by the matrix. The reason for thls behavior Is explained by the effectlve penetratbndepth of the beam, whlch Is shown to be only one or two partlcle dlameters when absorption by the matrlx Is strong.
Quantitative analyses performed with absorption spectrometry usually depend upon a linear relationship between band intensity and concentration. For diffuse reflectance (DR) spectrometry, Kubelka-Munk (K-M) theory indicates that linear plots of band intensity versus concentration should result when intensities are plotted as the K-M function 0003-2700/88/0360-2427$01.50/0
F(R) = (1- R)2/(2R) where R is the absolute diffuse reflectance of the analyte at infinite depth. F(R) is the ratio of the absorption coefficient, K, to the scattering coefficient, S. S = 2s, where s is the scattering coefficient per centimeter in the absence of absorption. K is equal to twice the Beel-Lambert law absorption coefficient, lz. For a nonscattering neat sample of path length b cm, having a transmittance of T
k = In (1/7‘)/b
(2)
Therefore, according to K-M theory for dilute samples in a scattering matrix
(3) where a is the absorptivity and c is the concentration of the analyte. Use of the Kubelka-Munk equation for quantitative analysis by diffuse reflectance spectrometry is common for measurements made in the ultraviolet, visible (I, 2), and mid(3)and far-infrared (4) regions of the spectrum, but not in the near-infrared (near-IR) region. As has been pointed out in several review articles (5-7), since Norris made the earliest reports on using near-IR reflectance spectrometry for the quantitative determination of components in agricultural products (8,9), almost all near-infrared DR spectra have been converted to log 1/R values prior to utilization in a program 0 1988 American Chemical Society
