Anal. Chem. 1988, 60, 1915-1920
1915
Refractive Index Gradient Detection of Femtomole Quantities of Polymers by Microbore Size-Exclusion Chromatography Darrell 0. Hancock and Robert E. Synovec* Center for Process Analytical Chemistry, Department of Chemistry, BG-IO,University of Washington, Seattle, Washington 98195
Unlversal and sensitlve detectors in high-performance liquid chromatography (HPLC) are generally considered mutually exclusive. Refractive index gradient (RIG) detection based upon Interferometry may overcome this technological barrier. A R I G detector has been constructed, studled, and appiled as a detector In microbore slze-exciusion chromatography of polymers. Eluting solute concentration profiles produce a RIG that deflects a singlemode dlode laser probe beam, producing a Sensitive measurement of chemical composition. Polymer detection Is a particularly dlfflcult test of the RIG detector slnce polydispersity leads to broadened chromatographic peaks, which limit RIG detectability. I n turn, the RIG detector sensitlvity to solute propertles holds excellent potentlai as a chemkai characterization tod. An effectlve R I detection limit of 4 X IO-' RI unit (3 X rms) is demonstrated. The mass detection limit (3 X nns) Is 540 pg (1.1 Mi)of 500000 g moT1 polystyrene Injected. This corresponded to a 0.9 ppm or 2.4 nM Injected concentration.
High-performance liquid chromatography (HPLC) is widely applied in most analytical laboratories. Much has been accomplished in HPLC methodology as outlined by Snyder and Kirkland (1). Recently, HPLC detector development has had vigorous activity (2-5),with detectors designed with a variety of important characteristics, i.e., sensitivity, selectivity or universality, robustness, and overall solute identification or characterization capability. Often, selectivity and sensitivity in HPLC detection are positively correlated, such as with absorbance or fluorescence detection, which leads to favorable detection limits and better detector applicability to samples of interest. Within this context, universal detection is quite popular since analyte derivatization is not required, while more sensitive methods, such as fluorescence, often require analyte derivatization. Unfortunately, universal detectors, such as a refractive index (RI) detector, generally have limited applicability when dilute samples are encountered, due to limited sensitivity. Ideally, a highly sensitive universal detector should be developed that does not require sample derivatization to enhance sensitivity. With universal detection, it is often difficult to utilize gradient elution, since the solvent contributes strongly to the base-line signal. The ideal universal detector would be highly resilient to solvent gradients. Thus, as Stolyhwo, Colin, and Guiochon pointed out (6),analogous to the flame ionization detector (FID) in gas chromatography, a sensitive universal detector for HPLC is not available. They developed a light-scattering (LS) detector for HPLC based upon an evaporative nebulization process (6, 7). The evporative LS has many favorable properties, yet unfortunately requires evaporation of the eluting solvent+olute mixture with preferential evaporation of the solvent. Further, the LS detector has moderate sensitivity compared to that of RI or absorbance detection (6). These factors may limit the overall universality of this LS detector, although excellent separations based upon gradients are feasible (7). A second approach to obtain "universal" detection in HPLC involves absorbance 0003-2700/88/0360-1915$01.50/0
detection in the UV range of 185-210 nm (8-10). Selected solvents were investigated to assess their transparency in this UV range. The list of solvents available as eluents for universal absorbance detection at 185-210 nm was quite useful yet may be limiting in many instances. A third approach to universal detection involves indirect solute detection (11-1 3). A property of the solvent is exclusively monitored by the detector, thus producing a "high" background signal. Displacement of the solvent (eluent) by a solute results in a net reduction in the solvent concentration as the solute/solvent mixture passes through the detector. This produces a negative detector response, i.e., an indirect solute signal. While indirect detection provides easy solute quantitation, solute characterization would require a second form of detection, i.e., another detector sensitive to some physical property of the solute(s) of interest. One realizes that RI detection remains the workhorse of everyday universal HPLC detectors. RI is a well understood chemical property that has led to new approaches to chemical analysis, i.e., complex sample characterization without analyte standards (14-17). RI detectors have been developed on the basis of four basic designs: refraction, reflection, interference, and the Christiansen effect (3). State of the art, in terms of RI detectability and detector robustness, varies for these designs. For capillary and microbore HPLC, Bornhop and Dovichi suggested an interesting refraction-type RI detector based upon a capillary flow cell (18).They applied the novel device in the reversed-phase microbore separation and RI detection of nanogram quantities of sugars (19).Further, they combined the capillary-flow-cell-based RI detector with simultaneous absorbance detection (20). When optimized, this capillary-flow-cell-based RI detector appears to be limited to about 3 X lo-' RI unit (3 X rms) as the limit of detection (LOD) (21). This detector is highly robust and simple to produce and maintain. The second type of RI detector, reflection-based,was optimized by Wilson and Yeung (22). They achieved a RI signal based upon the change in transmitted light at a liquid-glass interface, in accordance with Snell's and Fresnel's laws. Their device allowed for simultaneous RI, absorbance, and fluorescence detection with a 1-cm path length through the cell, in only about 1 pL of volume. RI detectability was comparable to that of the capillary-based RI detector (refraction-based), at about 1 X lo-' RI unit. The interference-type RI detector (23) yields an excellent LOD at 4 X RI unit, yet a relatively long path length, in the context of microbore or capillary HPLC, was required. The Christiansen effect RI detector has had few developments recently (3). Gradient applications with RI detection have been limited, since most consider this impractical if not impossible, yet gradients may be applied with RI detection by employing a volume delay loop for dual-cell RI detectors (24). Ideally, one would desire to utilize gradients in RI detection without requiring a volume delay loop, which contributes to solute peak broadening. Recently, Pawliszyn proposed an interesting approach to RI detection, based upon probing the RI gradient (25). The nature of the detected signal was attributed to a Schlieren effect. The sheath flow technique was applied to enhance 0 1988 Amerlcan Chemlcal Society
1916
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988 r w
outlet
n r,
I
U
:W LI
SYSTE M Figure 1. Experimental schematic for RIG detection of HPLC effluents: DL, diode laser; MO, microscope objective; P, polarizer (not required); C, flow cell (Figure 2); PD, photodiode detector; HPLC, summarized HPLC system; W, waste; WT, waveform generator; LI, bck-in amplifier; COMP, computer or recording device.
detector sensitivity. In subsequent studies of the detector properties (26),it was determined that the sensitivity was path length dependent; thus scaling down the device may sacrifice sensitivity. A predicted detection limit of 3 X RI unit was reported (26). A very interesting consequence of measuring the refractive index gradient (RIG) was realized Le., for linear eluent gradients, the RIG detector yields a rather small, constant, base-line offset (26). Thus, the RIG detector readily allows for eluent gradients, while providing excellent solute detectabilities. In this initial work on RIG detection (25,26), the nature of the detected signal was not fully described and approaches for improving the device were not addressed. Since interferometry is the most sensitive RI detection method available (23), one may expect to improve RIG detection similarly by an interferometric design. We have constructed and studied a sensitive RIG interferometric detector. Our device employs common path interferometry to provide a sensitive measure of probe beam deflection (27-29). Thus, a simple interferometric detector has been developed that measures RIG effects. The complete system is shown in Figure 1. The diode laser, flow cell, flow cell windows, and photodiode are the components that comprise the interferometric detector. Flow cell rotational angle and photodiode translational placement are two important experimental parameters to control. Injected femtomole quantities of polymers, separated by microbore size-exclusion HPLC, with RIG detection was demonstrated, corresponding to 4 X RI unit (3 X rms) detectability. A nonabsorbing wavelength for the eluent and the polymers (780 nm) was used. The device was relatively simple to construct and employ, although the shape of the observed solute response is atypical of conventional HPLC detectors, since the solute concentration gradient is monitored instead of the solute concentration. The RIG detector has many attributes that may lead one to believe that the ideal universal detector for HPLC is forthcoming. The RIG detection principle by an interferometric mechanism is examined experimentally, and subsequent detector performance is discussed.
EXPERIMENTAL SECTION The experimental system is shown in Figure 1. The 780-nm, 3-mW output from a single-mode diode laser (Physitec Corp., DL 25, Norfolk, MA) with a 10-m coherence length (minimum) was intensity-modulated at 20 kHz via a TTL waveform (Wavetek, Model 190, San Diego, CAI, which was also synchronized to a lock-in amplifier (Princeton Applied Research Corp.,Model 5204, Princeton, NJ). The modulated diode laser output (probe beam) was focused at a focal length of 60 cm to a spot size of 200 pm at a z-confguration cell (made in-house)via a micrmpe objective, f 16.85, that was specially designed for the diode laser system. For some initial work, the probe beam polarization was purified prior to reaching the flow cell by passing the beam through a polarizer (Karl Lambrecht, MGTYS8, Chicago, a)mounted and finely tuned by a high-precision rotational stage (Newport, Model
Figure 2. Flow cell model: I,, incident beam; I, transmitted beam; CW, cell window; CB, cell block (aluminum); no, eluent R I , n ( t ) , timedependent RI; inlet, from HPLC system; outlet, to waste.
471-A, Fountain Valley, CA). Probe beam polarization and polarization effects in general were not found to significantly affect the performance of the interferometric sensor. The flow cell (constructed from aluminum), configured as in Figure 2, had a cylindrical bore of 1.0-cm length X 800-pm i.d., thus producing a volume of 5.0 pL. The flow cell was also mounted on a highprecision rotational stage (Newport, Model 471-A) for front to back rotational fine tuning and on a high-precision x-y-z translational stage (Newport, 460-XYZ) for centering, and glass microscope slide cover-slips having a width of 0.20 mm were used as windows and were carefully glued at each end of the flow cell. For this work, the effective cell length is no greater than 800 pm, which was the flow cell inside diameter. In future designs, the flow cell length should be shortened with a concomitant volume reduction without sacrificing detection sensitivity, assuming similar hydrodynamic behavior (30,31). The entrance and exit tubing to the flow cell was highly flexible, yet sturdy, Teflon 1/16-in. 0.d. X 0.007in. i.d. (nominal). After the probe beam passed through the flow cell, it was imaged onto a photodiode (Hamamatsu, S1723-05, Hamamatsu City, Japan) with a 1 cm X 1cm active surface, which was mounted on a linear translational stage (Newport,460-X) for translation along the beam axis. The voltage output from the photodiode was sent to the lock-in amplifier to obtain the in-phase and subsequently demodulated analytical signal, synchronized by the waveform generator. The analytical signal was sent to either a personal computer (Il3M-XT, Armonk, NY) via a laboratory interface board (MetraByte, DASH-16, Taunton, MA) or a chart recorder (Houston Instruments, D-5000, Austin, TX) or simultaneously to both devices. The HPLC system applied in our studies consisted of methylene chloide as the eluent, which was delivered at 40 pL/min by a high-precision, high-pressure syringe pump (ISCO, LC-2600, Lincoln, NE) through an injection valve (Rheodyne, Model 7125, Cotati, CA) fitted with a 0.5-pL loop for subsequent sample introduction into the HPLC system. From the injection loop, the liquid was pumped through a 250 X 1.0 mm i.d., 300-A pore, 5-pm, CS microbore column (BrownleeLabs, RP 300, Santa Clara, CA), which performed adequately as a size-exclusion retention mechanism with spectrograde CH2C12as the eluent and polystyrenes (Polymer Laboratories, Amhurst, MA) as samples (32). As shown in Figure 2, the outlet of the microbore column passed into the flow cell via the inlet tubing, through the region intercepted by the probe beam, and continued out of the flow cell via the outlet tubing to waste. It is interesting to refer back to Figure 2 and the z-configuration cell. One initially can not imagine that a physical condition such as ordered flow from the inlet to the outlet exists in the flow cell. Indeed, at most flow rates encountered in conventional HPLC, about 0.3-2.0 mL/min, turbulence exists, and at any instant in time a homogeneous RI exists from one end of the flow cell to the other, and no RIG signal is observed. Rather dramatically, at lower flow rates of 20-300 pL/min, RIG signals are observed, suggesting ordered flow was present in this flow rate range. Furthermore, in the corners of the flow cell without an inlet or outlet tube, it is reasonable to suggest that stagnant eluent is trapped in eddy currents in an almost “equilibrium” condition (30). Yet, no hysteresis was observed due to solute “trapping” in our studies. The flow pattern shown in Figure 2 is suggested as a reasonable picture, consistent with experimental findings.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988 1917 0.06
F
..
I . .
q 0.00
(I)
2lu L F
-d
Flgure 3. Detection of R I gradient (model): I,, incident beam; I,, transmitted beam; F, direction of flow; CW, exit cell window; PD, photodiode; 8,, detected angle due to RIG gradient presence; 8, rotational tuning angle for flow cell; d, translational tuning distance for PD relative to CW. Furthermore, in this work the band broadening of polymer peaks, separated via microbore size-exlusion HPLC, was not adversely contributed to by the flow cell design or volume. In addition to the necessity for ordered flow through the flow cell to maintain solute RIG integrity, the interferometric RIG detector must be properly tuned to achieve good sensitivity. Examination of the tuning procedure is presented. A comparison was made between our RIG detector and a commercially available UV-vis absorbance detector for HPLC (ISCO, Model V4, Lincoln, NE) fitted with a 0.50-pL flow cell (ISCO, Series 0080-072) having a path length of 2 mm and operated at 270 nm for sensitive detection of the polystyrene samples. At this wavelength, 5000oO g mol-’ polystyrene has an extinction coefficient of 1.8 L g-’ cm-’, as measured on a UV-vis spectrometer. A 1-8 time constant was applied for both methods of chromatographic detection. This comparison was found to dramatically support the capability of the RIG detector. In order to fairly compare the two detectors, the path length difference must be considered; i.e., the absorbance detection would provide a detection limit a factor of 5 better if fitted with a 1-cm path length. The purpose of using the 0.5-pL flow cell with the absorbance detector was to compare peak broadening relative to the RIG detector, in the context of microbore HPLC of polymers. Fortunately, the volume of the RIG detector flow cell did not contribute significantly to peak band broadening; indeed, this would effectively reduce sensitivity (26). The microbore work was preceded by work with a conventional HPLC column, i.e., 250 X 4.6 mm i.d. Macrosphere, 300-Apore, 5-pm, C8 (Alltech, Deerfield, IL). Only the microbore results are shown for brevity. The selection of polymers (polystyrenes)as analytes was made in order to addreas a 2-fold problem. First, polymers are inherently polydisperse, meaning the eluted solutes produce peaks that are further broadened relative to monomeric species of identical molecular weight. This broadening reduces analyte sensitivity (25,26). Thus, detection of polymers with the RIG sensor becomes a worst-case scenario in order to truly “test” the RIG detector. Second, for many polymers an absorbing wavelength is not conveniently available, and analytical methods at trace levels are limited. The RIG detector is shown to be an attractive device for polymer detection in HPLC at nonabsorbing wavelengths.
RESULTS AND DISCUSSION The interferometric mechanism as visualized in Figure 3 was examined by two approaches. First, the photodiode lateral position, d , was kept constant, and the flow cell rotated, 8, by 1.1mrad increments, spanning 100 mrad, with the center of the range approximately where the probe beam was perpendicular to the cell windows. At each incremental setting the photodiode output was measured. The photodiode output data are essentially the base-line signals, B(8). An average base-line signal was calculated, B,and the relative difference calculated, Brel(8),a t each incremental setting
4 lu
.....-.
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-0.02-0.04-
U
0
10
20
30
40
50
ANGLE INCREMENT, mrad
Figure 4. Relative base-line signal, B,.&), as a function of angle Increment, 8, for flow cell rotation with the interferometric RIG detector. with the results for a portion of the 8 range traversed shown in Figure 4. The shape of B,l(O) versus 8 increment in Figure 4 is consistent with an Airy function, with a depth of modulation of about 10% and a periodicity of 35 mrad (33). By injection of a suitable model solute a t constant injected mass, flow rate (107 pL/min), and entrance tubing (28 cm of 0.007-in. i.d. Teflon) and detection of the RIG signal at each incremental angle, as for Figure 4,the relationship between Bm1(8) and the RIG signal was studied. As previously reported (25, 26), the RIG signal, 8d in Figure 3, was measured as the peak-to-peak response, labeled as VC, since a gradient of concentration is effectively measured as the maximum to minimum deflection angle
for a purely Gaussian peak, where Vi is the injected volume, Ci is the injected solute volume fraction, u,” is the volumetric variance of the solute elution profile, F is the radius of the probed cross section, no is the solvent RI, and dn/dC is a sensitivity factor describing the change in RI of a mixture as a function of the volume fractions o f t he species present. For a binary mixture with the solute at low volume fraction (14)
[-I
dn _ - (no2+ 2)’ n 2 - 1 dC 6n0 n,2 + 2
no2- 1 no2 + 2
(3)
where n, is the solute RI. VC was measured at each incremental angle, 8, thus VC(8) is compared to Brel(8). These results are pesented in Figure 5, where VC(8) and (B(8)-B)/10 are both plotted for approximately one full period in the Airy function (33). Referring back to eq 1, the quantity (B(8)B)/10 is plotted instead of B,,,(8) in order to simplify the plotting of Figure 5. Note that the VC(8) signal (peak-to-peak) is a positive value if the model solute RI is greater than solvent RI, as was the case for polystyrenes in CH2C12. It is clear from Figures 4 and 5 that there is a direct correlation between B,&3) and VC(8), with the conclusion that flow cell rotational position, acting as an etalon, must be considered in the performance of the RIG interferometric detector. A second approach to examining the interferometric mechanism requires keeping the flow cell rotation angle constant, with translation of the photodiode by incremental distances, d, along the laser beam axis. Incremental steps of 25-500 pm were taken, with the base-line signal as a function of distance B(d) measured, analogous to B(8) for flow cell rotation. Simultaneously VC(d) was measured at each incremental distance for the same model solute as in VC(8) measurements. The results are shown in Figure 6, where
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988
0.4
h
30
50
40
ANGLE INCREMENT, mrad Flgure 5. Relative base-line signal calculated as (5(0) - 8 ) / l O (W) and RIG signal VC(0) (0)for flow cell rotation.
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4
a 2
(3
I
cn
m
E
0
6
TIME,& Figure 7. Microbore sizesxclusion HPLC separation of 46 ng of injected 500 000 g mol-' polystyrene (2.8 min) and 5 1 ng of injected 9000 g mol-' polystyrene (4.0 min): A, R I G detection; B. UV-vis absorbance detection (270 nm). -11 2
'
1
'
4
6
8
10
12
14
DISTANCE, d, mm Figure 6. RIG signal VC(d) (0) as a function of photodiode lateral displacement from the flow cell exit window with the interferometric RIG detector. V C ( d ) is plotted as a function of the lateral translational distance, d, in millimeters. B(d) has not been plotted in Figure 6 for clarity, although B(d)was correlated to VC(d) in the same way B(0) was correlated to VC(0) in Figure 5. Clearly, translational positioning of the photodiode relative to the flow cell (Figure 5) produces an Airy function (33) analogous to Figures 4 and 5, where flow cell rotation was examined. It is interesting to note in Figure 6 that RIG detection sensitivity is optimized a t 3.5 and 13.5 mm, where large jumps in sensitivity were observed. The periodicity of the large jumps is about 20 mm. Underlying these large jumps in sensitivity is a VC(d) versus distance ( d ) dependence that is less pronounced. The periodicity of the less pronounced sensitivity positions is about 8 mm. One would desire to work at an optimum in sensitivity, which occurs at either 3.5- or 13.5" lateral displacement between the flow cell exit window and the photodiode. In practice, satisfactory RIG detection sensitivity is obtained even if one works some distance from the optimum, but prior knowledge of the periodicity of a given RIG interferometric detector, for both flow cell rotation and photodiode lateral displacement, is essential for rapid dayto-day tuning. It is interesting to compare the microbore size-exclusion chromatograms obtained with a UV-vis absorbance detector (commercial, 270 nm) and with the RIG detector (Figure 1). The chromatograms are shown in Figure 7, where 500000 g mol-' polystyrene has been separated from 9000 g mol-' polystyrene. Quite impressively, the RIG detector, Figure 7A, provides a marked improvement in detectability over the
conventional UV-vis absorbance detector, Figure 7B. Figure 7A appears to have less high-frequency noise and a larger low-frequency noise component than Figure 7B. This appearance is misleading. Note in Figure 7, parts A and B, that the signal is relative in each case and also on different "sensitivity" scales, distorting the appearance of the base-line noise. One must consider the signal-to-noise ratio, which for the same injected sample (two solutes) favored the RIG detection (A) over the UV-vis absorbance detector (B) by a factor of 8 for the earlier eluting solute (500000 g mol-' polystyrene). Note that the UV-vis absorbance detector is functioning quite well, with a LOD (3 X rms) of 7.2 x lo4 AU. Furthermore, the absorbance detector is being operated a t a favorable wavelength for the solute, as indicated by the high extinction coefficient of 1.8 L g-' cm-'. Thus, for weakly absorbing or nonabsorbing solutes, the RIG detector offers substantially improved detection over absorbance detection and slightly better results for even the highly absorbing solutes, as shown in Figure 7. The trade-off between the two methods occurs when one considers the variance of the solute elution profile in the context of the detector physical characteristics, since RIG detection sensitivity decreases as peak width increases (25,26,34).This will be discussed shortly. While the RIG detector produces a signal that is atypical for most HPLC detectors, simple integration algorithms may be applied by the analyst, if desired, to arrive at a more conventional shape and possibly improve detectability simultaneously via the integration (35, 36). Furthermore, peak identification and quantitation will be more straightforward once the RIG-detected chromatogram is integrated. We have applied the gradient detector to a separation mixture of five polystyrenes, with conventional size-exclusion HPLC,with similar results as shown in Figure 7. Clearly, the RIG detector is quite promising for the analysis of many analytes, even if an absorbing wavelength is available, as was the case in Figure 7. One could imagine that the RIG detector is far superior in
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988 2oo
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dz
1919
Table I. Comparison of Predicted Concentration Gradient Signal Ratios t o Experimentally Obtained Concentration Gradient Signal Ratios Polystvrene
v)
w
100
P I-
170000 680 000 34 500
4 Lu n 100
0
200
INJECTED MASS, ng Figure 8. Calibration plot for quantitative analysis of polymers using microbore size-exclwlon HPLC and RIG detection: A, 500 000 g mor' polystyrene; 8 , 9000 g mol-' polystyrene.
the detection of species when an absorbing wavelength is not readily available or is limited by experimental considerations, e.g., when sample derivatization is not practical or possible. The RIG detector provided calibration curves for the polystyrenes separated in Figure 7. Calibration curves are shown in Figure 8 where the RIG signal, VC, in arbitrary units, has been plotted as a function of the injected analyte mass for two polystyrene standards labeled A and B. Linearity is obtained for about 2 orders of magnitude, Le., from the LOD to about 100 ng injected, at which point the interferometric signal begins to level off. The LOD was calculated as 3 times the standard deviation of the base-line noise (3 X rms). For 500 000 g mol-' polystyrene, the LOD with the RIG detector coupled with microbore HPLC was 540 pg injected, or 1.1 X mol. Alternatively, the injected concentration for the 500000 g mol-' polystyrene at the LOD was 0.9 ppm or 2.4 nM. The reproducibility of the signal, in the context of HPLC detection, appears to be limited to the consistency of the peak widths. Any slight overloading of the column will result in a changing peak shape, generally broadened, which subsequently results in reduced sensitivity in RIG detection. This relates to the concept of sensitivity, which depends upon the RIG-detected signal VC described by eq 2. For two solutes labeled 1 and 2, detected under the same chromatographic conditions, i.e., volumetric flow rate and injection volume Ci,l a,? -VC1 - - (dn/dC)I --
VC2
(dn/dC)zCi,2 0,,1'
(4)
where dn/dC, Ci, and u,2 are defined as in eq 2 and 3. Comparison of sensitivies based upon known injected solute volume fractions and measured peak broadening is readily obtained by eq 4. It is clear that by maintaining low peak broadening, i.e., small ,:u one enhances the detected RIG signal. Experimental evidence in support of this idea is given in Table I. For three polystyrene standards, at known injected volume fraction Ci, the peak variance 0,2 of chromatographically retained peaks was measured by the UV-vis absorbance detector, both by width-at-half-height and width-at-base-line measurements. It was assumed that dn/dC is the same for the polystyrenes, which is reasonable for this work (37). Both approaches for measuring:u gave similar results. Basically, one may predict from eq 4, via the W-vis u: data, the relative responses of the solutes, a priori, that one should obtain with the RIG detector. With 170 000 g mol-' polystyrene used as the reference point for the predictions, the ratios Ci,z and u,,?/ u,,? were calculated and, finally, multiplied together to yield the predicted concentration gradient ratio VC1/VCz as in eq 4. This was compared to the concentration gradient
1.00 1.58 2.12
i.od
1.od
1.od
0.40 0.20
0.63 0.42
0.65 (0.03) 0.48 (0.02)
Nominal values; note that each polymer had nearly the same polydispersity. Peak variance ratio measured from UV-vis absorbance data. Known injected volume fraction ratio. Predicted concentration gradient signal ratio, calculated according to eq 4, assuming each polymer has the same RI as explained in text. e Experimentally measured concentration gradient signal ratio average of three trials, assuming each polymer has the same RI as explained in text, and the standard deviation (s). '170000 g mol-' polystyrene is the reference point for these ratios, i.e., u ~ , ' C~j,,', and VC..
ratios measured from experimental data obtained for three trials with the same species and chromatographic system by RIG detection. The comparison between predicted and experimentally obtained concentration gradient ratios was quite good, as compiled in Table I. The primary ramification of eq 4 and Table I is to stress that band broadening, i.e., u?, should be minimized prior to RIG detection in order to optimize detection sensitivity. The effective RI LOD for 500 000 g mol-' polystyrene was quite impressive. Had the concentration level at the LOD been measured via more conventional RI detction approaches, the inferred RI LOD is 4 X RI unit for 500000 g mol-' polystyrene in Figure 7. What we have calculated is the effective RI detection limit, assuming the solute had been detected with a conventional RI detection mechanism (3). The detection limit, An, was readily calculated (14) given n, = 1.600 (polystyrene, nominal) (37), no = 1.424 (CHzC12),and the signal-to-noise ratio of a RIG signal obtained for a known injected volume fraction of 500 000 g mol-' polystyrene in CH2ClZ,in the linear region of the calibration curve shown in Figure 8. Alternatively, the sheath flow technique has been successfully applied with RIG detection (26). In this approach, and in ours, careful expansion of the chromatographic effluent is important to maintain a sharp, yet enhanced, concentration gradient in the flow cell, which may help to explain the inferred angular deflection LOD. The 13 term in eq 2 indicates the dependence on careful effluent expansion. The r3 effect was discussed in earlier reports (25,26)but not put into the quantitative expression relating to the RIG signal VC (eq 2). The improved detection limit obtained by our system is strong evidence that a z-configuration flow cell is well-suited for RIG detection. Clearly, the observed interferometric signal with the device has ramifications, in general, for flow cell design and application in analytical chemistry. Alternatively, a position-sensitive detector (PSD) may be used to detect the RIG signal, as has been demonstrated (26,34). A PSD produces a more straightforward measure of beam deflection (38) as compared to the interferometric mechanism described here, but the reported detection limit is roughly a factor of 10 poorer with the PSD technique (26) than with the interferometric mechanism. The RIG detector, with further improvements, may well be the universal detector of choice for HPLC, with a present LOD of 4 X RI unit (3 X rms). Note that this LOD depends not only on the RI differences but also on the presentation of the solute-solvent mixture via a gradient to the interferometric detector, which depends upon the chromatography and flow cell properties. As inferred by eq 4, any
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Anal. Chem. 1988, 60, 1920-1924
improvement in the separation efficiency, reducing uv2,will result in an improved detection limit. This will require a concurrent decrease in the flow cell volume to limit band broadening at detection. Our flow cell volume was just small enough to limit broadening in the microbore HPLC of polymers, since the flow cell volume was about a factor of 10 less than detected solute peak volumes. Since the RIG signal is quite sensitive to the eluting solute peak width, further investigations have been directed toward exploiting this dependence to more sensitively probe changes in polymer samples, as may occur in processing conditions. The novel device is extremely simple to construct and maintain, and thus, may be quite useful for process analysis applications as well as routine laboratory work.
(9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27)
ACKNOWLEDGMENT D.O.H. and R.E.S. thank L. Burgess and M. Schurr for helpful discussions concerning the interferometric mechanism. Registry No. Polystyrene, 9003-53-6.
(28) (29) (30) (31)
LITERATURE CITED
(32) (33)
(1) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed.; Wley-Interscience: New York. 1979. (2) Yeung, E. S.; Synovec, R. E. Anal. Chem. 1986, 58, 1237A-1256A. (3) Yeung, E. S. In chemlcel Analysis, Detectors for Liquid chromatoga phy, Vol. 89; Elving, P. J., Winefordner, J. D., Eds.; Wiley-Interscience: New York, 1986. (4) Munk, M. N. I n Liquld ChromatographyDetectors; Vickrey, T. M.. Ed.; Dekker: New York, 1983. (5) Scott, R. P. W. Liquid Chromatography Detectors, Elsevier: Amsterdam, 1977. (6) Stolyhwo, A.; Colin, H.; Guiochon, G. J . Chromatogr. 1983, 265, 1-18. (7) Stolyhwo, A,; Colin, H.; Guiochon, G. Anal. Chem. 1985, 5 7 , 1342-1354. (8) Berry, V. V. J . Chromatogr. 1980, 199, 219-238.
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(34) (35) (36) (37) (38)
Berry, V. V. J . Chromatogr. 1982, 236, 279-296. VanDerWal, S.; Snyder, L. R. J . Chromatogr. 1983, 255, 463-474. Small, H.; Miller, T. E. Anal. Chem. 1982, 5 4 , 462-469. Bobbin, D. R.; Yeung, E. S. Anal. Chem. 1984, 56, 1577-1581. Mho, S. I.; Yeung, E. S. Anal. Chem. 1085, 57, 2253-2256. Synovec, R. E.; Yeung, E. S. Anal. Chem. 1983, 55, 1599-1603. Synovec, R. E.: Yeung, E. S. J . Chromatogr. 1984, 283, 183-190. Synovec, R. E.; Yeung, E. S. Anal. Chem. 1984, 56, 1452-1457. Synovec, R. E.; Yeung, E. S. J . Chromatogr. Sci. 1984, 23, 214-221. Bornhop, D. J.; Dovichi, N. J. Anal. Chem. 1986, 58, 504-505. Bornhop, D. J.; Nolan, T. G.; Dovichi, N. J. J . Chromatogr. 1987, 384, 181-187. Bornhop, D. J.; Dovichi, N. J. Anal. Chem. 1087, 59, 1632-1636. Synovec, R. E. Anal. Chem. 1087, 59, 2877-2884. Wilson, S. A,; Yeung, E. S. Anal. Chem. 1085, 5 7 , 2611-2614. Woodruff, S. D.: Yeung, E. S. Anal. Chem. 1982, 5 4 , 2124-2125. Woodruff, S. D.; Yeung, E. S. J . Chromatogr. 1983, 260, 363-369. Pawliszyn, J. Anal. Chem. 1086, 58, 243-246. Pawliszyn, J. Anal. Chem. 1988, 58, 3207-3215. Dandridge, A.; Miles, R. 0.; Giallorenzi, T. G. Electron. Lett. 1980, 16, 948-949. Dandridge, A. Appl. Opt. 1981, 2 0 , 2336-2337. Dandridge, A,; Tveten, A. B. Appl. Opt. 1081, 2 0 , 2337-2339. Kachei, V.; Menke, E. I n Flow Cytometty and Sorting, Hydrodynamic Properties of Flow Cytomefric Instruments;Melamed, M. R., Mullaney, P. F., Mendelson, M. L., Eds.; Wiley: New York, 1979; Chapter 3. Lighthill, J. Waves in fluids; Cambridge University Press: Cambridge, England, 1978; pp 286-298. Renn, C. N.; Synovec, R. E. Anal. Chem. 1988, 6 0 , 200-204. Hecht, E.; Zajac, A. Optics; Addison-Wesley: Reading, MA, 1979; Chapter 9, pp 301-307. Pawliszyn, J. J . Liq. Chromatogr. 1087, 10, 3377-3392. Synovec, R. E.; Yeung, E. S. Anal. Chem. 1985, 57, 2162-2167. Synovec. R. E.; Yeung, E. S. Anal. Chem. 1988, 58. 2093-2095. Merck Index, 10th ed.; Merck: Rahway, NJ, 1983; No. 8732. Renn, C. N.; Synovec. R. E. Anal. Chem. 1988, 6 0 , 1188-1193.
RECEIVED for review March 8,1988. Accepted May 16,1988. D.O.H. and R.E.S. thank the NSF Center for Process Analytical Chemistry for support of this work (Project Number 86-2).
Determination of Carboxylic Acids by Isotope Dilution Gas Chromatography/Fourier Transform Infrared Spectroscopy Edwin S. Olson,* John W. Diehl, and Michael L. Froehlich
University of North Dakota Energy and Mineral Research Center, Box 8213, University Station, Grand Forks, North Dakota 58202 A method has been developed for determlnlng carboxylic acids by Isotope dllutlon with ‘*O-enrIched carboxylic aclds and measuremsnt of the Isotope ratlo In methyl esters of the anaiyte and standard mlxture by caplllary gas chromatography/Fourler transform Infrared (GC/FTIR) spectroscopy. The dlfference In the carbonyl absorptlon maxima for the analyte and standard esters allowed separate absorbance chromatograms to be reconstructed by lntegratlon over two narrow frequency ranges in each analytehtandard GC/FTIR spectrum. Area ratios obtained from the absorbance reconstructed chromatograms were plotted versus concentration ratios to give a nonlinear cailbratlon plot, which was expressed as a thlrd-order polynomial by least-squares polynomlal fitting. The method was more accurate than a G U M S method developed wlth the same standards, analytes, and range of concentrations. The Isotope dilutlon GC/FTIR method was appiled to the quantitative analysis of aqueous mixtures of coal oxidation products.
The quantitative analysis of mixtures of carboxylic acids resulting from the oxidation of coals presents a challenge to
the analytical chemist. These products consist mainly of aliphatic and aromatic di- and polycarboxylic acids, which are highly soluble in the aqueous reaction medium. Inorganic compounds are often present and may react with derivatizing reagents. Determinations of carboxylic acids and other highly polar organic compounds usually require addition of isotope enriched internal standards to obtain accurate results, because even highly polar homologous standards are neither extracted from aqueous solutions nor derivatized reproducibly or to the same degree as the analytes. A deuterium isotope gas chromatography/mass spectrometry (GC/MS) method was used for analysis of the products of coal oxidation with ruthenium tetraoxide (1,2). This method was accurate when the standard possessed four or five deuterium atoms per molecule (3)but less accurate than desired in determinations with standards that possessed only two deuterium atoms per molecule. However, the lack of possible sites for deuterium substitution in aromatic polycarboxylic acids and the unfortunate ease with which hydrogens are exchanged in carboxylic acids with acidic hydrogen on carbon (e.g., malonic acid) make deuterium isotope dilution GC/MS difficult to apply to these compounds. Although 13C-enriched aliphatic dicarboxylic acids are not difficult to prepare, they are relatively expensive, and 13C-
0003-2700/88/0360-1920$01.50/00 1988 American Chemical Society
