Moving belt interface with spray deposition for liquid chromatography

The moving belt interface provides potentially one of the more versatile approaches to coupling LC to MS in that it permits operation of the mass spec...
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Anal. Chem. 1983, 55, 1745-1752

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Moving Belt Interface with Spray Deposition for Liquid Chromatography/Mass Spectrometry M. J. Hayes, E. P. Lantkmayer,' Paul Vouros,* and B. L. Karger* Institute of Chemical Analysis and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115

J. M. McGuire United States Environmental Protection Agency, Athens Environmental Research Laboratory, Athens, Georgia 30605

A systematic study of the chromatograptmlc performance of a movlng belt Interface for LCIMS was conducted. For thls work a speclally deslgned nebullzer was constructed for deposltlon of the effluent from the LC column onto the movlng belt. The role of various; parameters of the nebulizer (e.g., gas flow rate and temperature) on chromatographic performance, as measured by the second moment of the generated chromatographlc profiles, was examlned. The Influence of belt speed on peak varltrnce was also determlned. On the basis of these studies, the LC separation wlth normal bore (4.6 mm 1.d.) columns was obtained under high-performance condltlons along wlth mass spectral analyses of complex mixtures of substances. Successful operatlon wlth moblle phases of high water conitent was also achieved. I n addition, detectlon llmlts as low ais 40 pg were measured In the E1 mode wlth polynuclear irromatlc hydrocarbons, with linear dynamlc ranges of at least 4 orders of magnitude. These studies demonstrate the dlllty of the movlng belt Interface for on-line high-performance! LC/MS.

The interest in on-line IAC/MShas been increasing in recent years, as a basic understanding of the characteristics of the various interfacing methaids has developed. Among the most promising interface approaches are the moving belt ( I ) and direct liquid introduction by use of chemical ( 2 ) and thermospray (3) ionization methods. Applications have been demonstrated in a variety of areas (4-7), and the power of the technique has been enhalnced by using MS/MS (8). The general development of the moving belt interface has progressed a t a somewhat slower rate than the direct liquid interface, in part due to problems associated with reversedphase solvents and appllicability to quantitative analysis. Significant progress has,however, recently been made by using a continuous segmented flow extractor system (9, I O ) , spray deposition ( I l ) , and microbore LC/MS (12,13). A review has recently been published (14). The moving belt interface provides potentially one of the more versatile approaches to coupling LC! to MS in that it permits operation of thle mass spectrometer in both the electron impact (EI) and chemical ionization (CI) modes. Moreover, this interface lends itself to use in conjunction with other methods of sample vaporization beyond the current commercial mode of flash vaporization. Indeed, the generation of SIMS spectra has been demonstrated with the moving belt approach (15, 16). The use of the moving belt interface in conjunction with fast atom bombardment ionization for on-line LC/MS has also been demonstrated (17). The purpose of this palper is to explore approaches to the operation of the belt interface that yield good chromatographic Present address: Institute fur Analytische Chemie, Technical University, Graz, Austria. 0003-2700/83/0355-1745$0 1S O / Q

fidelity with normal bore columns. Data have been obtained on the degree of band broadening and the applicability of the belt interface to quantitative analysis. In particular, the primary role of the mode of sample deposition onto the belt has been studied by using a spray deposition approach modified from the nebulizer initially developed for the moving wire flame ionization detector (18)and subsequently employed in a moving belt interface (11).Extracolumn band broadening contributions of the system have been minimized to a level that high-performance LC/MS can be carried out in both the normal and the reversed-phase modes. Moreover, detection limits in the picogram range have been achieved for polynuclear aromatic hydrocarbons. Preliminary results of this work have previously been reported (19).

EXPERIMENTAL SECTION A Finnigan (Sunnyvale,CA) 4000 mass spectrometer equipped with a moving belt, LC/MS interface with Kapton belts was used in all experiments. The mass spectrometer was operated in the E1 mode with an indicated source temperature of 250 "C. For the belt drive mechanism, a variable speed motor was substituted for the interchangeable fixed speed motor normally employed with the Finnigan interface. The method for transfer of the chromatographic effluent to the belt surface was based on spray deposition. The spray device, shown in Figure 1,consisted of (A) the liquid transfer line, made from 8 cm X 1/16 in. o.d., 0.01 in. id., stainless steel tubing (Alltech Associates, Deerfield IL). The bottom 10 mm of the tube was tapered to a point, approximately 0.3 mm wide, in order to form the spray orifice. The tube was inserted coaxially into a 1.5 cm long outer sheath (E, in Figure 1) of 1/4 in. o.d., 1/8 in. i.d. Pyrex glass tubing drawn to a 6 mm long taper with a 0.6 mm i.d. opening at the tip. The assembly was held together by a stainless steel union (B) that was threaded into the belt housing. The device was mounted on the interface by drilling a hole on the housing, centered over the belt, at 30" from the vertical. In use, the height of both the glass and steel portions could be independently adjusted to yield the desired spray conditions. The spray was formed at the orifice by the high shear forces generated by a flow of nitrogen gas. Throughout this work the nebulizing gas for forming the spray was controlled by a low-pressure regulator. For less volatile polar solvents, the gas was heated immediately upstream from the sprayer with a heater (Hotwatt, Inc., Danvers, MA) placed immediately before the spray apparatus and powered by a variable transformer. The gas temperature entering the sprayer was approximately 80 "C for the higher water content mobile phases. The connections between the heater and the spray adapter were wrapped with heating tape in order to maintain the gas temperature relatively constant. (Additional information on the spray device is available from the authors.) For experimental studies where numerous repeat injections were required, plug injections were made in the following manner. A Waters (Milford, MA) M6000A pump was employed with a Valco (Houston, TX) air-actuatedsix-port injector connected to a 100-wL tube. This arrangement provided reproducible reference solute peaks that could be easily characterized with a UV detector. Samples were injeckd at short intervals to minimize the problems 0 1983 American Chemical Society

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ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983

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Plgure 1. Diabram of spray deposition device: (A) transfer line from LC and spray orifice ‘/re in. o.d., 0.007 in. i.d. stainless steel (SS); (6) SS body ot spray device; (C) retalnlng cap; (D) O-ring seal; (E) Pyrex tip to form gas spray; (F) movlng belt; (G) gas inlet. Details can be found in the Experimental Section.

of instrument drift that can occur in the mass spectrometer over extended periods of time. Liquid chromatography was performed with a Varian (Palo Alto,CA) 5000 ternary liquid chromatograph and a Valco six-port air-actuated injection valve. A Varian UV 50 variable-wavelength detector was used for the LC/UV studies. The chromatographic columns were either a 10 cm X 4.6 mm, 7.5 pm Zorbax BP-C8 (Du Pont, Wilmington, DE) packed in our laboratory by using conventional slurry packing techniques or a 20 cm X 4.6 mm, 5 pm nitro bonded phase Nucleosil column (Macherey-Nagel, Germany). Solvents were HPLC grade, purchased from Fisher Scientific (Medford, MA) and degassed by using vacuum with ultrasonic agitation. Reagents and chemicals were purchased from Aldrich (Milwaukee, WI) and used as received. A gas chromatographic procedure was employed as an external standard for quantitative LC/MS experimedts carried out over a period of several hours. This approach assured that the observed changes in signal intensity were due to the belt interface and not to changes in the sensitivity of the ion source itself. A 2 m long packed column of 3% OV-l/Supelcoport (Supelco, State College Park, PA) was used and 4 pL of 5a-androstane (20 ng/pL) was injected in order to monitor system sensitivity. Measurements of solute transfer efficiency to the belt were performed by first spraying a peak from the chromatographic column onto the belt and then comparing that peak area with one obtained when depositing the same mass of sample of the belt with a syringe. The syringe was assumed to provide 100% transfer of solute to the belt.

RESULTS AND DISCUSSION The goal of this work was to study the chromatographic and quantitative aspects of the moving belt interface and to make modifications that would improve the overall utility of the system. The belt interface can be thought of as consisting of four basic steps each of which can affect chromatographic performance. The first is the transfer or deposition step, where the overall goal is to apply the column effluent in a uniform thin film onto the belt. The second step is the evaporation of the solvent remaining on the belt with an infrared heater, sometimes in conjunction with a slight vacuum of about 100 torr. The solute then passes into the vacuum lock system in the third step. If the solute is immobilized in a thin film on

Flgure 2. Comparison of solvent deposition modes: (A) continuous flow, (B) spray deposition. Insets show typical flow patterns for each method. Flgure shows separation of fluorene, pyrene, triphenylene, and benzo[a]pyrene: 20 cm X 4.6 mm, Nucleosil5 pm nitro bonded phase, 5 % methylene chloride/hexane, 1 mL/min.

the belt as it enters the vacuum system, then transport within the vacuum system should, in principle, have little affect on chromatographic fidelity. The desorption of the sample from the belt and into the ion source is the fourth step. This process should have no influence on chromatographic efficiency, because the rate of diffusion of vaporized solute out of this region will be fast relative to the time scale of the eluting chromatographic profiles. The evaporation step, however, may have an effect on the quantitative response of the interface. These four regions of the interface are not independent of one another; thus, a problem in one step can lead to additional problems in subsequent steps. We have found the deposition step to be critical in obtaining good chromatographic performance and quantitation. It has been found necessary to change the conventional deposition system, and we have adopted a spray deposition approach modified from that of others (11,18). The influence that the deposition step can have on the performance of the interface is illustrated in Figure 2, which shows a comparison of the conventional method of flowing the effluent onto the belt in a continuous stream vs. spray deposition. All experimental conditions were the same except for the mode of sample deposition onto the belt. The total ion chromatograms are based on the normal phase separation of a simple mixture of polynuclear aromatic hydrocarbons (see figure caption for conditions). Both chromatograms provide good mass spectra of the compounds studied. Figure 2A shows the result of effluent being placed on the belt with the commercially available glass tip. It can be seen that broadened and distorted peaks with a number of spikes are obtained. Improved peak shape can be achieved, especially when using nonaqueous solvents, by appropiate optimization of the transfer step; however, Figure 2A does emphasize potential problems. The inset to Figure 2A shows one of the causes of the poor chromatographic behavior. A large volume of liquid was often present on the belt when using direct deposition; at times droplets would form in contact with the glass tip and belt. These droplets would become especially pronounced as mobile phases of high polarity were used, as in reversed-phase LC. This behavior clearly provides for a zone of intermixing that would be expected to cause contributions to band broadening. Beyond the mixing effect, the droplets released from the large liquid zone were deleterious for several reasons. First, it was difficult to evaporate the solvent completely before entering the vacuum lock system. At times, particularly for polar solvents, the possibility existed for some solvent to

ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983

remain on the belt until the flash evaporation step. This excess solvent could cause pressure surges in the ion source resulting in large spikeri on the peaks and the base line. A second type of spiking was caused by nonuniform distribution of sample droplets on the belt during thle evaporation step that would result in irregularities and distortion on the solute peaks, as seen in Figure 2A. Interestingly, others have seen spiking of peaks when a moving wire was dip-coated with solution (see Figure 4 of ref 18). In view of the above, we recognized that a more efficient solvent evaporation anld sample deposition procedure was necessary. Using the nebulizer system described in the Experimental Section, we achieved total ion1 chromatograms as shown in Figure 2B. Narrowed peak widths and profiles free of spikes were observed. Dispersion of tlhe LC effluent into a fine mist provided an efficient evaporation step. The design of the sprayer was arrived at after testing several different configurations. We found that the dimensions of the orifice through which the liquid flows should be minimized in order to prevent liquid from accumulating on the glass tip and in order to allow th,e formation of thle smallest possible droplets. Further adjustments revealed that the end of the steel transfer line should extend approximately 0.5 mm past the end of the glass. M[oreover, the glass tip should be 4-8 mm above the belt surface. Finally, by using a 60" angle between the spray tip and the belt, droplet formation on the belt, behind the tip, was minimized. These droplets were formed as a result of the conical shape of the spray pattern and, when present, were found to contriblute significantly to the variance of the system. With the encouraging results of Figure 2, we then decided to examine in detail the spray deposition procedure. Repeat injections were made by using plug injections of 5 pL of either acridine, 5a-androstane, or indenol into a 'LOO-pL tube to yield reference peaks for determination of the s,ystem performance. For these studies, it was necessary to employ statistical central moments in order to calculate peak area, retention time, and variance. Because the peaks are not fully symmetrical, large errors would result, particularly in the second and higher moments, if Gaussian peaks were assumed. The moments are calculated by the following equation:

:ch(t - E)''

n t h central moment = M , = -

Ch

(1)

where M,, is the nth moment, h is the peak height from base line at time t , and f is average time. A computer program was written to perform the above calculations directly from the Finnigan INCOS data system. The average, standard deviation, and percent standard deviation were also calculated far each moment. (Further details can be obtained upon request.) To obtain the net variance (second moment) contribution of the MS detector system, the variance determined from the interface system was subtracted from reference values obtained with a UV detector. The reference peaks throughout this study were generated in a similar manner as those in the MS experiment using the 100-pL tube system described in the Experimental Section. The reference variance from the UV detector signal was 1600 pL2.

SAMPILE DEPOSITION As discussed above, the spray depositioin step is of primary importance in obtaining good chromatographic fidelity, including peak shape, variance, area, and reproducibility. For this study, our strategy was to fix the dimensions and position of the tip above the belt and vary the slpray conditions by means of the nitrogen gas flow rate and temperature. In this approach, the spray could easily be altered without major changes in the spray device. Two aspects of the deposition

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Figure 3. Effect of gas pressure on MS peak variance (pL2)and relatlve peak area by using spray deposition, injections of acridine in methylene chloride, 1 mL/min: (A) effect of spray gas pressure on variance; (B) effect of spray gas pressure on area.

step were analyzed on this basis: the gas pressure, or flow of nitrogen gas, arid the flow rate of the eluent from the LC column. In addition, we explored the effect of the type of mobile phase employed on the spray deposition conditions. Gas Flow Rate. In the first set of experiments, we examined the role of nitrogen gas flow on chromatographic performance with a fixed liquid flow rate. With a particular tip position that appeared to work well for a fairly broad range of solvent conditions (6 mm above the belt), the gas temperature was set to provide evaporation of a significant fraction of solvent with a gas pressure of 5 psi. The gas pressure was then fine tuned for optimum performance. The interface was operated at a fixed belt speed of 3.4 cm/s and a desorption temperature of 240 "C. Sample plugs of 5 pL of acridine in methylene chloride (20 ng/pL) were injected into a flowing stream of methylene chloride at 1 mL/min and subsequently sprayed onto the moving belt. Ten replicate samples were injected for each pressure and the peak variance (in pL2) and area were determined by averaging the values from each sample. As mentioned above, the MS peak variance was determined by subtracting the measured sample variance of the MS interface from the measured variance of the UV detector. Figure 3A shows a plot of MS peak variance against gas pressure. The MS variance decreases considerably with an increase in gas pressure, reaching a plateau of 4-5 psi. The decrease in MS peak variance with increasing gas pressure or flow rate can be explained in terms of the formation of smaller spray droplets, yielding more efficient evaporation of the solvent. A thinner film on the belt is produced resulting in less mixing of the solute and reduced beading. Given the large change in variance with gas pressure, we concluded that this parameter is very significant in optimization. Figure 3B shows a plot of relative peak area for acridine against gas pressure. In this measurement, all peaks are normalized to the largest area. Peak area (Figure 3B) increases by more than 2-fold as gas pressure is increased, again leveling off at 4-5 psi. Thus, optimized variance and response appear to occur under the same spray conditions, further emphasizing the importance of gas pressure on performance. One possible cause for the lower peak area at lower gas pressure in Figure 3B is the wetter spray. Some losses from the belt due to splattering occurred when the belt surface appeared to have excessive liquid. In addition, liquid flowed to the underside of the belt when the surface was wet. Thus, there appears to be a relationship between the wetness of the spray and the solute response from the moving belt. Figure 4 illustrates the changes in peak width and shape for single ion monitoring of plugs of acridine when the gas pressure was varied. The first peak was generated by using 0.5 psi of gas pressure and each peak generated at 1.0 psi higher pressure so that the last peak was obtained with 8.5 psi. The peaks at low gas pressure are split and show considerably higher variance than the peaks obtained at higher

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Flgure 5. Effect of flow rate on MS variance (pL2)and relative peak area: (A) change in variance with flow rate, (X) indicates the variance for 2.4 mL/min after optimization of spray pressure; (6) change in area with flow rate. Injections of acridine into methylene chloride. Flgure 4. Comparison of peak shape wlth changes in spray pressure: inJections of acridine into 50/50 (v/v) water/methanol at 0.66 mL/min; peak 1 at 0.5 psi, other peaks incremented by 1 psi: peak 9 at 8.5 psi; gas temperature 50 O C .

gas pressure. Peak splitting is believed, in part, to be a result of the buildup of drops of mobile phase on the tip a t low gas pressures. These droplets could be sporadically swept onto the belt to yield the observed doublet peak shape. Moreover, such unmixed zones could also contribute to extracolumn band broadening. It is also possible to have too high a gas pressure at which point gas velocities cause the belt to vibrate and lose sample/solvent. This loss of liquid can result in both reduced sample transfer efficiency (loss in peak area) and irregular peak shape. This behavior occurred at higher pressures than those shown in Figures 3 and 4. In this case, more efficient vaporization of the solvent would result from an increase in temperature a t satisfactorily lower gas pressure. Liquid Flow Rate. The second phase in the examination of the deposition step was to monitor the effect of changing the liquid flow rate from the LC column. The belt speed was again set a 3.4 cm/s, the desorption heater was set a t 240 "C, and plug injections were made with acridine. The spray gas pressure was maintained at 5 psi, and the liquid flow rate of methylene chloride varied from 0.6 to 2.4 mL/min. No signal was obtained for injections made below 0.6 mL/min since all of the solvent was vaporized before it could reach the belt and little or no deposition appeared to occur. Above 2.4 mL/min the peak shape became badly distorted and the ion source pressure began to rise significantly due to large amounts of solvent entering the vacuum system. Figure 5A shows a plot of net variance from the MS interface vs. liquid flow rate. The variance increased slowly at flows between 0.6 and 1.5 mL/min, beyond which large changes in variance occurred. The greater amount of liquid on the belt at the high liquid flow rates caused increased mixing and variance, in agreement with the results previously found for the effect of gas flow rate on variance. Another effect that occurred a t high flow rates was that the excess solvent on the belt struck the vacuum slit before evaporation was complete. This created an unswept liquid zone that could contribute to the variance and result in solvent spikes in the chromatogram. The poor results at high liquid flow could be significantly improved by increasing the gas pressure to 10 psi, as indicated by the lower point at 2.4 mL/min. Clearly, gas pressure and temperature can compensate for changes in mobile phase flow rate. This result reinforces the point that optimization of the spray conditions can, in large measure, be handled by adjustment of the nebulizing gas, while maintaining nozzle dimensions and position constant. Ultimate optimization, however, may require attention to nozzle dimensions and

position above the belt, as well. Figure 5B shows a plot of relative area vs. liquid flow rate. Maximum response is obtained in the range of 0.7-1 mL/min for a gas pressure of 5 psi. The results in this figure are consistent with the previous trends of relative area plotted against gas pressure in Figure 3B. Above 1 mL/min, peak area in Figure 5B is rapidly reduced with increasing flow rate. The higher the liquid flow rate in this case the wetter the spray. We have previously suggested several factors that could lower response when a wetter spray is used, including potential overflowing from a wet belt. It is interesting to note that in ref 18 a plot of detector response liquid flow rate for spray deposition on the moving wire detector was shown. A loss in signal a t high liquid flow rate was observed in ref 18 and in the present work. A wet spray is clearly detrimental to transport devices. As noted above, the lower peak area at 0.6 mL/min in Figure 5B appears to be a consequence of too dry a spray. In this case it is possible that some portion of the sample could adhere to the spray tip as the sample is dried when exiting the tip. In addition, if the droplets from the nebulization are completely dry before reaching the belt surface, then the sample may not fully adhere to the belt. Indeed, below 0.6 mL/min no signal could be observed for these experimental conditions. Similar relationships to those shown in Figures 3 and 5 hold true for different solvent systems including systems with high water content. One can obtain good performance for a given system by altering the gas temperature and pressure. An example of this is shown in Figure 6 in the isocratic separation of four phenols on a C8 reversed-phase column using a mobile phase of 74.5/24.5/1 (v/v/v) water/acetonitrile/acetic acid at 0.5 mL/min. Both UV and MS (total ion) chromatograms are shown. Even with this high water content mobile phase and in the presence of acetic acid, on-line mass chromatograms maintaining good chromatographic peak shape and efficiency are obtained with normal bore columns. Indeed only slight losses in resolution are observed on the MS total ion chromatograms. In this example, efficient evaporation required heating the nitrogen gas to roughly 80 OC. For all mobile phases, we have found in practice that if the liquid from the spray tip is observed to form a film of fine droplets that evaporate rapidly after deposition on the belt, good results will be obtained. By rapid evaporation we mean that a wetted area will be observed on the belt extending approximately 0.5-2 cm from the point of deposition. Once a reasonably good spray is achieved, fine adjustment can be made by varying the pressure. For the case of large gradients (e.g., 100% water to 100% organic), it may be necessary to vary the temperature of the nebulizing gas during the course of a run in order to maintain optimum chromatographic efficiency. For less difficult cases, e.g., 40% methanol water to 100% methanol, one can generally obtain adequate control

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ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983

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Flgure 8. Comparison of LC/UV and LC/MS profiles under high water content mobile phase condiions: belt speed 3.4 cmls, spray pressure 10 psi, gas temperature 80 OC, 15 cm X 4.6 mm, 7.5 gm Zorbax BPC8 column, 74.5/24.5/ 1 (vlvlv) water/aceionitrile/acetic acid, flow 0.5 mL/min; (1) benzyl alcohol, (2) p-nttroben2yl alcohol, (3) 1-indenol, (4) cinnamyl alcohol.

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IBELT S P E E D We also conducted a brief study of the effect of belt speed on the performance of the system. We selected spray conditions that gave good deposition at a belt speed of 3.0 cm/s, and a desorption temperature of 240 OC A series of ten plug injections was made for each of several belt speeds while holding all other condlitions constant. The plug injections consisted of 5 gL of a 25 ng/pL solutbon of 5a-androstane injected into the open tube described previously. The solvent was methylene chloride at a flow rate of 0.6 mL/min, sprayed onto the belt by using: unheated gas at a pressure of 6 psi. Figure 7A shows that the variance from the MS interface changes with belt speed reaching a relatively broad minimum at the 3.0-4.5 cm/s range. The increase in variance a t low speeds is believed to occur because the sample was deposited on a small portion of thle belt. The belt appeared wet, resulting in remixing of the peak before the solvent was removed. At high speeds (>4.9 cm/s), the eluent struck the vacuum lock before the solvent could evaporate. As described before, this effect may cause liquid to build up at the slit entrance causing spreading of solute. Presumably, even higher belt speeds may result in base line and peak spikes similar to those shown in Figure 2A. The effect of belt speed on peak area can be seen in Figure 7B, in which relative area is seen to increase to a maximum with belt speed. If the speed was too low, the solute could start vaporizing before proper positionin,g of the sample band with respect to the ion source, resulting: in a loss in sample. In addition, as mentioned above, the wetness of the belt at low belt speeds may cause sample loss. With the use of spray deposition, the role of belt speed should be less important in maintaining chromatographic fidelity than with continuous liquid deposition, since the latter would result in more solvent being on the belt for a given

Figure 7. Chromatographic effect of belt speed with spray deposltlon, injectlons of 5a-androstane in methylene chlorlde 1 mL/mln: (A) effect of belt speed on MS variance; (B) effect of belt speed on relative peak area. Belt speed is in cm/s.

mobile phase flow rate. The precise location of the optimum belt speed for the spray deposition approach can vary with the particular solvent type and deposition conditions, but due to the broad nature of the minimum, speed settings between 3.0 and 4.5 cm/s will generally give good performance. A belt speed of 3.4 cm/s was used throughout this work.

CHROMATOGRAPHIC PERFORMANCE From the results above, we used the optimum values obtained for extracolumn variance contributions by the interface and calculated the losses in resolution that would be expected to occur for representative chromatographic systems. The variance of a peak eluting from a given chromatographic column was calculated by use of eq 2 Ucol

=

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where Vois the retention volume of an unretained peak, k' is the capacity factor, and N is the number of theoretical plates. This value was then used in eq 3 to find the expected loss in resolution. uMS represents the extracolumn variance contributed by the interface itself

Ai?=[,_

1

]x 100 (3) (1 + .MS2/%012)1'2 where AR is the percent loss in resolution, uMSis the variance contribution from MS, and uc,l is the variance of chromatographic peak at column exit. Table IA summarizes calculated losses in resolution that would be incurred for high-resolution columns for various k ' values. In this example we have selected an efficient column

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Table I. Calculation of Resolution Loss due to the Moving Belt Interface

___

A. Theoretical Section

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column: 7.5 pm, Zorbax BP-C8, 1 5 cm, 9000 plates; mobile phase: 30/70 (v/v) water/acetonitrile, 0.6 mL/min solute

k'

% resolution loss

pyrene p-terphenyl anthracene coronene

1.2 2.0 2.2 3.5

13.7 12.4 11.1 6.7

with a reduced plate height of three. I t is apparent that the predicted losses in resolution are relatively small for It'values above one. These predictions were based on measurements of the artificially generated profiles used for the evaluation of the effect of gas pressure, belt speed, and flow rate in which the variance contribution from the MS was assumed equal to 500 pL2. To confirm these calculated results, we measured the losses in resolution incurred when using an actual chromatographic system. In this example, we used an isocratic separation of the four polynuclear aromatic hydrocarbons (PNA's), listed in Table IB, on the Zorbax BP-C8 column previously described, with a mobile phase of 30/70 (v/v) water/acetonitrile. The resolution loss (eq 3) was determined from the measured extracolumn variance based on the intercept of a plot of variance against retention time squared (20). The actual losses in resolution (Table IB) agree well with the predicted values in Table IA and the resolution loss is small a t It'values above 1. Even for the more difficult case of 75% water (Figure 6), chromatographic resolution is maintained very well (an average loss on resolution of only 10% is estimated for each solute pair). The losses in Figure 6 appear to be due to a small amount of tailing at the base of the peak. In summary, these results show that for normal bore columns, high-performance chromatography with high water content mobile phases can be achieved by HPLC/MS, using the moving belt interface.

LC/MS APPLICATIONS Following optimization of the variables outlined above, we next examined several chromatographic systems to demonstrate the utility of the spray deposition method for on-line LC/MS applications. The results are shown in the chromatograms below for both normal- and reversed-phase systems. Figure 8 shows normal-phase gradient elution chrornatography of ten PNA's using hexane/methylene chloride a t 1 mL/min on a bonded phase column with nitro group functionality. Gradient and column conditions are described in the figure. There is excellent correlation between the MS total ion and UV chromatograms. The four peaks F, G, H, and I in the reconstructed ion chromatogram are distinguished, as in the UV chromatogram. Close examination of the two chromatograms shows that the width at half height is similar for both cases. Any loss in resolution in the reconstructed ion chromatogram is due almost entirely to a small amount of tailing, as discussed above. Figure 8C shows the additional power of the mass spectrometer over UV as a detector in its ability to deconvolute overlapping peaks having different

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Flgure 8. Comparison of LC/UV and LC/MS profiles for normal-phase gradient chromatography: belt speed 3.4 cm/s, spray pressure 5 psi, 3as temperature 35 O C , 20 cm X 4.6 mm, Nucleosil 5 pm nitro bonded ,base, hexane to 8% methylene chioride/hexane in 15 min, 1 mL/min; :a)anthracene, (b) p-terphenyl, (c)pyrene, (d) 1,2:4,5-dibenzopyrene, :e) benzo[b]fluoranthene,(f) indeno[ 1,2,3-cd]pyrene,(9)picene, (h) 3enzo[g]perylene, (i) truxene, (j)1,2:3,4-dibenzanthra~ene.

fragmentation characteristics. Thus, peaks F, G, H, and I can be individually isolated on the basis of single ion monitoring. (The spectra of compounds F and H have the same base peak.) The value of the spray deposition method is perhaps best realized when considering its applicability to reversed-phase HPLC. I t should be emphasized that the use of aqueous mobile phases including gradients, in normal bore columns, Imcompasses a major portion of current HPLC applications. As noted earlier (Figure 6), use of the spray deposition technique allows retention of chromatographic efficiency in an isocratic system of high water content. This is further illustrated in Figure 9 which shows a more complex mixture of PNA's. In this case gradient elution starting with a mobile phase high in water is necessary to achieve resolution of the compounds in the mixture (65/36 (v/v) water/acetonitrile t o 100% acetonitrile at 1mL/min on a Cs bonded phase column). As in Figure 8, a good correspondence between the UV chromatogram and the reconstructed ion chromatogram is observed. Note especially that the reconstructed ion chromatogram can discriminate peaks G, H, and I which greatly

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Figure 9. Comparison of LC/UV and LC/MS profiles for reversedphase gradient chromatography, belt speed 3.4 cm/s, spray ptessure 8 psi, as temperature 85 OC, 15 cm X 4.6 mim, 7.5 pm Zorbax BP48 column 65/35 (v/v) water/acetonitrile to 100% acetonitrlle in 20 min, flow 1 mL/min; (a) phenanthrene, (b) anthracene, (c) fluoranthene, (d) pyrene, (e) chrysene, (f) unknown, (9) benzo[b]fluoranthene, (h) benzo[k]fluoranthene, (11) benzo[a Ipyrene, (i) benzo[a ]anthracene, (k) indeno[ 1,2,3-cd]pyrene, (I) unknown.

overlap one another. Figures 6,8, and 9 clearly demonstrate that the spray deposition approach can be used for analysis of complex mixtures >withnormal bore columns under highperformance separatiion conditions in the gradient elution mode.

QUANTITATIVE ANALYSIS Having demonstrated that chromatolgraphic fidelity could be maintained by using the spray deposition approach with the moving belt system, we next turned to the problem of quantitative analysis. We used three PNA's for this study: 1,2:4,5-dibenzopyrene, l,Z-benzanthraicene, and 1,2:3,4-dibenzanthracene. The compounds were separated on the nitro bonded phase column in the normal-phase mode with 8% methylene chloride in hexane a t a flow rate of 1 mL/min. Figure 10 shows log--log calibration plots for the three PNA's in terms of peak area vs. concentration of injected sample. The injection volume' was 10 pL, and the MS was operated in the selected ion mode for each of the three molecular ions. A linear dynamic range of 4 orders of magnitude is observed for the f i s t compound, 1,24,5-dibenzopjnene (the actual linear range is probably greater than this). Moreover, for this compound, detection of 40 pg in the 110fiL injected was obtained with a S I N ratio of 2.5:l (see Figure 10 inset). The dilution from chromatographic travel through the 20-cm column was estimated to be 15-fold. Thus, an even lower detection limit may be possible by minimizing this column dilution (21). (Of course the other solutes undergo more dilution since their k' values are higher than 1,2:4,5-dibenzopyrene.) Diveqgence of the calibration curves from a

i

2

"

' ' 25' " ' 28 LOG C O N C E N T R A T I O V

36

4c

(pgiul)

Flgure 10. Calibration curves obtained by E1 (selected ion monitoring of molecular ions) for three PNA's as indicated. Chromatography was performed on 20 cm X 4.6 mm, Nucleosil 5 pm nitro bonded phase, 8% methylene chloride/hexane (vlv), 1 mL/min. Inset shows S I N for the lowest concentration of 1,2:4,5dibenzopyrene. Concentration is picograms injected on-column.

common slope of unity in Figure 10 is due to the nonzero intercepts of the area vs. concentration plots. Regression coefficients greater than 0.997 were obtained for the later calibration plots, confirming linearity. The results in Figure 10 demonstrate the potential of the moving belt interface as a trace analytical LC detector. This potential is in addition to the power of the mass spectrometer as a qualitative analytical tool. While the thermal stability and E1 fragmentation pattern of the PNA's are favorable to trace level detection, subnanogram detection is seen to be achievable. An issue related to the detection limits is the transfer efficiency onto the belt surface which, if poor, could compromise trace level detection. The transfer efficiency was measured by comparing the peak area (zeroth moment) for the same mass of solute placed onto the belt, first by spraying and then by direct application with a syringe. All experimental conditions were the same, including the width of the plug profiles. The sample transfer efficiency for indenol using 74.5/24.5/1 (v/v/v) water/acetonitrile/acetic acid at 0.5 mL/min was measured to be 70%. For other solutes using nonaqueous systems, the transfer efficiency was found to be as high as 95% due to the milder spray conditions required. These high recoveries are supported by the low limit of detection obtained in the calibration curve (Figure 10). An examination of the reproducibility of the area measurements taken from the experiments on flow rate and belt speed resulted in relative standard deviations of 3-6%, for the average of ten repetitions of a particular measurement. These results, including the calibration curve, are without an internal standard and we thus absolute measurements of area. The use of internal standards, particularly of labeled analogues, would undoubtedly improve these values.

CONCLUSIONS In this work we have examined the moving belt interface system for LC/MS and have demonstrated that the key to obtaining good chromatographic performance when utilizing normal bore columns is the deposition step. Marked improvement has been seen in chromatographic efficiency and peak fidelity by using spray deposition with a variety of mobile phases and flow rates. A system is presented that is practical for both qualitative and quantitative analysis using on-line high-performance LC/MS with normal bore columns. Future work will be devoted to a further investigation of high water content mobile phases and the possible use of the spray deposition approach with microbore columns. Finally, it needs to be recognized that the flash evaporation process is only one

1752

Anal. Chern. 1983, 55, 1752-1760

of many possible approaches to vaporization from a solid surface. As mentioned in the introduction, methods such as SIMS, FAB,etc. show real promise and may eventually allow the moving belt LC/MS interface to become practical for virtually any compound class. We have conducted this study for ultimate application of LC/MS to environmental problems. As an example, in the treatment of drinking water, unexpected substances are formed from the interaction of disinfection chemicals with unknown trace organic compounds present in the untreated water. In order to understand the disinfection chemistry and to assess the potentially adverse health effects, the unknown chemicals must be identified both before and after treatment. Many of these compounds are highly polar (22) and cannot be analyzed in a cost effective way by techniques such as gas chromatography/MS. LC/MS would appear to be of potential value in this analysis.

LITERATURE CITED (1) Eckers, C.; Games, D. E.; Lewis, E.; Nagaraja Rao, K. R.; Rossiter, M.; Weerasinghe, N. C. A. Adv. Mass Spectrom. 1980, 86,1396. (2) Henion, J. D. Adv. Mass Spectrom. 1980, 86, 1241. (3) Biakeiy, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750-754. (4) Cairns, T.; Seigmund, E. G.; Doose, G. M. Anal. Chem. 1982, 5 4 , 953-957. (5) Henion, J. D.; Mayiin, G. A. 6iomed. Mass Spectrom. 1980, 7 , 115-12 1. (6) McFadden, W. H.; Bradford, D. C. J . Chromatogr. Scl. 1979, 17, 5 18-522. (7) Games, D. E.; Lewis, E. 6iomed. Mass Spectrom. 1980, 7 ,433-436. (8) Henion, J. D.; Thomson, B. A.; Dawson, P. H. Anal. Chem. 1982, 5 4 , 45 1-456. (9) Karger, B. L.; Kirby, D. P.; Vouros, P.; Foitz, R. L.; Hidy, B. Anal. Chem. 1979, 51,2324-2328.

(IO) Kirby, D. P.; Vouros, P.; Karger, B. L.; Hidy B.; Peterson, B. J. Chro-

matogr. 1981, 203, 139-152. (11) Smith, R. D.; Johnson, A. L. Anal. Chem. 1981, 53, 739-740. (12) Games, D. E.; Lant, M. S.;Westwood, S.A.; Cocksedge, M. J.; Evans, N.; Williamson, J.; Woodhaii, B. J. 6iomed. Mass Spectrom. 1982, 9 , 215-224. (13) Games, D. E.; Hewlins, M. J.; Westwood, S.A.; Morgan, D. J. J . Chromatogr. 1982, 250, 62-67. (14) Aicock, N. J.; et ai. J . Chromatogr. 1982, 251, 165-174. (15) Benninghoven, A.; Eicke, A.; Junack M.; Sichtermann, W. Org. Mass Spectrom. 1980, 15, 450-453. (18) Smith, R. D.; Burger, J. E.; Johnson, A. Anal. Chem. 1981, 53. 1603-1611. (17) Dobberstein, P.; Korte, G.; Meyerhoff, G.; Pesch, R. I n t . J. Mass Spectrom. Ion Phys. 1983, 46, 185-188. (18) van Dijk, J. H. J. Chromatogr. Scl. 1972, IO, 31-34. (19) Lankmayer, E. P.; Hayes, M. J.; Karger, 8. L.; Vouros, P.; McGuire, J. M. Int. J. Mass Spectrom. Ion Phys. 1983, 4 6 , 177-160. (20) Kutner, W.; Debowski, J.; Kemuia, W. J. Chromatogr. 1981, 218, 45-50. (21) Karger, B. L.; Martin, M.; Gulochon, G. Anal. Chem. 1974, 46, 1640-1647. (22) Christman, R . F.; Johnson, J. D.; Norwood, N. L.; Liao, W. T.; Hass, J. R.; Pfaender, F. K.; Webb, M. R.; Bobenrieth, M. J. PB 81-181952; NTIS: Springfield, VA.

RECEIVED for review February 7,1983. Accepted May 1,1983. The research described in this article has been funded by the U.S. Environmental Protection Agency under assistance agreement number CR807325-02 to Northeastern University. The contents of this article do not necessarily reflect the views and policies of the agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsment or recommendation for use. This is Contribution No. 156 from the Institute of Chemical Analysis.

Gradient Elution Chromatography with Microbore Columns H. E. Schwartz and B. L. Karger* Institute of Chemical Analysis and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115 P. Kucera Pharmaceutical Research Products Section, Quality Control Department, Hoffman-LaRoche, Inc., N u t l e y , N e w Jersey 07110

This paper deals wlth the development of practical approaches to mlcrobore column gradlent elution chromatography through the modlflcatlon of equlpment currently available for normal bore columns. Key to the deslgn Is the use of varlous hlgh-pressure, low volume mixers. The Influence of poor mixing on base llne nolse Is first examlned, and it Is shown that the column can amplify the slgnal due to unmlxed zones, partlcularly If one or more components of the moblle phase possess some UV absorbance. By use of the amplitude of the base llne nolse as a relative measure of mlxer performance, two static mlxers, a specially designed dynamlc mlxer and a combined dynamWstatlc mlxer, are examlned. I n additlon, response and delay volumes as well as retentlon and peak area preclslon are measured for each mixer. The mlcrovolume dynamlc mlxer Is shown to yield small delay (26 pL) and response (46 hL) volumes with low nolse characterlstlcs. By use of the dynamlc mlxer alone or in combination wlth a statlc mlxer, hlgh sensltlvlty mlcrobore separations of peptldes, proteins, and phenols are shown.

In the past several years there has been increasing interest in the use of microbore columns for HPLC (1-12). Packed

columns of 1to 2 mm i.d. are now commercially available, and there is potential for the possible use of open tubular columns, if appropriate instrument design can be achieved. Microbore columns as a consequence of low mobile phase flow rates have potential for coupling with specific detectors such as mass spectrometry (2, 13-15), flame photometry (16), Fourier transform IR (17), and electrochemical (18, 19). Solvent economy is another reason for the interest in microbore HPLC. It is worth noting that this economic factor can be significant when synthesized or expensive agents that permit high selectivity (e.g., chiral species) are used. In addition, the increasing low level detection of substances with highly sensitive detectors require improved purification of solvents and reagents. These added purification steps will inevitably increase the expense of such solvents. Another potential advantage of microbore columns is the improved mass detectability relative to normal bore columns (e.g., 4.6 mm i.d.), as a consequence of the low dilution in the narrow bore columns. In order to realize the full potential of microbore columns with respect to mass detectability, it will be necessary to use columns with small particle diameters, packed with high efficiency. Detectors which permit full realization of detection at low noise levels along with negligible contributions to band broadening will also be required.

0003-2700/83/0355-1752$01.50/00 1983 American Chemical Society