Anal. Chem. 1982, 54, 2583-2586
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Effects of Pressure, Temperature, and Solvent Composition in Analysis by Direct Liquid Introduction Liquid ChromatographyIMass Spectrometry Robert D. Voyksner,"" Carol E. Parker, and J. Ronald Hass National Institute of Envlrcnmental Health Sciences, P.0. Box 12233, Research Triangle Park, North Carolina 27709
Maurice M. Bursey Department of Chemlstty, Universlty of North Carolina, Chapel Hill, North Carolina 275 14
The dependence of slgiiai Intensity and degree of fragmentation In dlrect llquld IntriDduction liquid chromatographylmass spectrometry (DLI-LC/IMS) on source pressure, solvent composltlon, and temperature Is demonstrated. The results show that the sample response varies with pressure and that optimum source pressure varies wlth solvent composltlon. Thls causes some dlfflcuity m i malntalnlng optlmai operating condltions In a gradlent elution analysis. Varying the solvent composltlon has no affect on protonated molecular ion sensltlvlty; rather It changes the amount of fragmentatlon observed. The extent of frsgmentatlon also depends greatly on temperature. The use of solvent cluster Ions and source pressure as a rapld method of optimlratlon of the DLI probe position is discussed.
We present data from a study concerning the effects of pressure, temperature, and solvent composition on the spectrum of benzoic acid by LC/MS for two common solvent systems in reversed-phase liquid chromatography.
The increasing use of direct liquid iintroduction liquid chromatography/mass spectrometry (DLI-LC/MS) (1)raises fundamental questions about the effects of interface tuning and solvent choice on sensitivity and fragmentation. The effects of the source parameters on chemical ionization mass spectrometry have been investigated (2-4) but the DLI parameters such as probe position, jet length, and solvent choice on the mass spectra have yet to be determined. Most experimental procedures involve the use of a tuning solution to optimize probe position and jet length for lbest sensitivity (5), If this tuning procedure can be replaced by optimizing for the solvent cluster ions or source pressure, the setup time can be reduced. If conditions are optimized by monitoring source pressure, then the effect; of pressure changes on sensitivity becomes an important question in determining the reproducibility of an analysis. Most DLI work reported uses a source temperature of 250-300 "C (6, 7).This high temperature ensures solvent and analyte vaporization, but the question arises as to whether lower temperatures can improve the spectra. The effects of temperature on ion intensity and fragmentation have not been established for reversed-phase solvents in LC/MS, as has been done with isobutane and methane chemical ionization (2,8). Solvent compositions for LC analysis have been based on the ability to provide the best separation (in terms of resolution and speed of analysis). Little consideration has been given to whether or not the best solvent for LC provides the best sensitivity for the MS analysis. There is, however, little information reported on the question of sensitivity changes with solvent manipulation and there are few choices in solvent composition that perform the analysis satisfactorily.
EXPERIMENTAL SECTION The acetonitrile and methanol were Burdick and Jackson (Muskegon, MI), distilled in glass quality; the water was J. T. Baker (Phillipsburg,NJ) analyzed HPLC grade. The benzoic acid was purchased from Aldrich Chemical (Metuchen,NJ), 99% pure. Solvent mixtures of 5/95,20/80,40/60,60/40,80/20, and 100/0 (volume/volume) were used for both acetonitrilefwater and methanol/water. The concentration of benzoic acid was 1 mg/mL in each mixture. The organics were filtered through a 0.5-pm fiiter and the water through a 0.45-pm filter (Millipore,Bedford, MA). Each solution was sonicated to remove dissolved gas which can cause unstable jetting of the LC/MS interface. The HPLC system was modeled by a Waters 6000A pump (Waters Associates, Milford, MA) delivering a continuous flow of benzoic acid solute. Since a continuous flow of benzoic acid was used, the injector, column, and UV detector were omitted. The pump was operated at a flow rate of 1 mL/min. The effluent entered the DLI-LC/MS probe interface (Hewlett-Packard,Palo Alto, CA) where a 1:lOO split allowed approximately 1% to enter the mass spectrometer. The droplets forming the jet developed through a 5 pm diameter orifice and were vaporized in the desolvation chamber attached to a modified Finnigan CI source previously described (9,lO). The mass analysis was carried out with a tandem quadrupole mass spectrometer (Extranuclear Laboratories, Pittsburgh, PA). The first quadrupole in the instrument was set in RF mode, where it acted as a focusing device rather than a mass discriminator. The positive ions passing the quadrupole rods were detected by an off-axis multiplier and recorded on a UV oscillograph. Typical operating procedures included visually checking the jet for stability, adjusting the jet length to 3-3.5 cm in length, and then observing that the total ion current for the cluster ions remained constant. The distance from the probe tip to the source block was monitored by the use of a calibrated sleeve that fits around the probe and rested against the vacuum housing. The source pressure was measured by an MKS Baratron capacitance manometer, type 220B (MKS Instruments, Burlington,MA). The operating pressure ranged between 0.1 torr and 0.7 torr. After the probe was in the vacuum system, ion current and pressure stability were checked, since fluctuations could occur if the orifice became plugged or if the HPLC pump was not operating properly. The normal operating parameters on the tandem quadrupole were as follows: electron energy, 100 V; emission current, 1 mA extractor, 4 V; and quadrupole 1 in RF mode. The source temperature for most experiments was 190-210 O C . For the temperature study the temperature ranged from 140 to 340 O C . The spectra were recorded (mass range of 40-150) at a 5 amu/s scan rate.
Graduate student, Department of Chemistry, University of North Carolina, Chapel Hilll, NC 27514.
RESULTS AND DISCUSSION Pressure Studies. Changes in source pressure can be brought about by changing the jet length or probe position
0003-2700/82/0354-2583$0 1.25/0
0 1982 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982 100
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Table I. List of the Optimum Source Pressure and Probe Distance (Measured from the Ion Exit Aperature of the Source to the Probe Tip) for Various Volume Ratios of AcetonitrilelWater and Methanol/Water volume ratio
source pressure, mtorr
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in the desolvation chamber. The latter technique was investigated only because of the problems in controlling and measuring jet length changes. Figure 1shows how the probe position can change the source pressure. When the probe is close to the source, it occupies most of the desolvation chamber, making gas escape difficult, and a higher pressure results. The optimum distance of the probe from the source and corresponding source pressure, for a fixed jet length of 3.5 cm, for various methanol/water and acetonitrile/water mixtures is given in Table I. These values were obtained by observing the maximum signal for the (M + H)+ of benzoic acid. The probe distance from the source does not ensure reproducible operating conditions since the jet length is difficult to reproduce from day to day. Variations in the orifice's diameter, wind currents, and changes in solvent cause variation in the jet length and volume of solution sprayed in the source. If the jet length or volume varies, then the source pressure will change and the probe will no longer be at the position that gives the best signal for the analyte. For this reason source pressure was chosen as the method for optimization. The plot of intensity of (M + H)+ and (M + H - H20)+of benzoic acid vs. pressure at various solvent mixtures for acetonitrile/water (Figure 2) and methanol/water (Figure 3) contains information about operational characteristics of the LC/MS interface. The plots show that the most intense signal
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Figure 2. The ion intenslties of (M H)+ and (M H - H20)+ of benzoic acid vs. source pressure at different solvent ratios of acetonitrlWwater.
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Figure 3. The ion intenslties of (M 4- H)' and (M H - H,O)+ of benzoic acid vs. source pressure at different methanol/wate;ratios.
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for the (M H)+ of benzoic acid occurs at higher pressures as the water concentration increases. Secondly, these plots show that the maximum response for the (M + H)+ ion of benzoic acid ( m / z 123) is at a lower source pressure than the maximum for the (M + H - HzO)+fragment ( m / z 105). The extent of fragmentation depends upon pressure too. For example, for an 80/20 acetonitrile/water mixture the maximum response for (M + H)+is at 0.175 torr, resulting in a (M + H - H,O)+/(M H)+ ratio of 1.8. On the other hand, if the response is maximized for (M + H - HzO)+,the optimum pressure is 0.250 torr, giving a (M + H - HzO)+/(M + H)+ ratio of 2.4. It is interesting to note that as the concentration of water increases there is less difference between the maximum response pressures for (M H)+ and (M + H - H20)+. Solvent Studies. The extent of fragmentation also varies with solvent composition. The ratio of the absolute intensities of (M H - H20)+and (M H)+ from Figures 2 and 3 at the point where (M H)+ maximizes, plotted against solvent composition, produces Figure 4. For both methanol/water and acetonitrile/water the (M + H - H,O)+/(M + H)+ ratio
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982 500[?H.CN
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Flgure 4. The (M H H,C))+/(M H)+ intensty ratio at the maximum (M H)+ signal of benzoic acid, from Figure 2 and 3, vs. solvent ratio for methanol/water and acetonitrlWwater.
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decreases as water concentration increases with the interface optimized on the (M IH)+ ion at each solvent ratio. These variations in fragmentation do not follow the theoretical correlation with proton affinity (PA) of the reagent. The PA's of acetonitrile, methanol, and water are 187, 184, and 167 kcal/mol, respectively (11). In chemical ionization the degree of fragmentation is expected to increase with increasing PA difference between sample and reagent (12). This is in fact observed in other classical CI experiments where the CI reagent is introduced into the source as a gas from a heated reservoir and where the Elample is introduced by direct probe. For the DLI-LC/MS results, there are two possible explanations for the increasing fragmentation trend with decreasing PA differences. First, water, because of its viscosity and surface cohesion, tends to form larger droplets than the organic solvents. This size difference, combined with the high heat of vaporization of water, allows longer droplet lifetimes (13) which could result in condensation of the nebulized liquid on the desolvation chamber. Secondly, the differences in the extent of fragmentation observed in the two cases can also be explained in terms of AH", for the dominant reagent ion and the sample (14,15).If different reagent ions than (H30)+, (CH3CNH)+, and (CHgOH2)+-for example, (H20),H+, (CH3CN),H+,and (CH,C)H),H+-are responsible for sample protonation, then the ordler of AH",values would be different. Changes in AH",up to 15-20 kcal/mol h,ave been reported as the number of molecules M or M' attached to a core ion MH+ changes (16,17).Different AHorvalues for protonation from different cluster ions deposit different amounts of internal energy in the sample, resulting in an unexpected extent of fragmentation, observed. Solvent composition becomes an important consideration when performing a gradient elution analysis. The plots of intensity vs. pressure at various solvent mixtures (Figures 2 and 3) indicate that during a gradient elution there is a variation in signal intensity if the DLI proberemains in one fixed position. For example, assume that a desired separation can be achieved with a solvent program of 20/80 to 80/20 acetonitrile/water. If the instrument is optimized at either 80/20or 20/80 solvent ratio, there is a loss in sensitivity by approximately a factor of 2 at the opposite extremes. If the instrument is tuned for the middle of the solvent program, the loss at the extremes is observed to be reduced by a factor of 1/1.3. The decrease in relative intensity, once recognized, should pose no problem in a gradient elution analysis. The absolute sensitivity, when the DLI interface is optimized at each solvent composition,of the protonated molecular ion of benzoic acid remains1 essentially constant over the entire
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Flgure 5. The pressure at which the optimum slgnal is observed for the (M -I-H)+ ion of benzoic acid at various solvent ratios for methanol/water and acetonitriie/water.
solvent composition range for both acetonitrile/water and methanol/water, Furthermore, benzoic acid shows the same response in methanol/water as in acetonitrile/water. In this case the choice of solvent composition seems to have no effect on the response for the analyte. Optimization. The optimization of the LC/MS interface in regard to pressure can be achieved easily by using the relationship between pressure and the maximum intensity of the analyte signal. Figure 5 shows how the pressure that gives the most intense (M H)+ signal varies with solvent composition for common solvents. With this information the best operating pressure for various solvent ratios or for different gradient elutions can be quickly determined. The observation that most high mass solvent clusters ( m / z 83, 95, 108 for acetonitrile/water and m / z 65, 79, 101 for methanol/water) maximize together with the analyte suggests their use in tuning. Optimization at a fixed solvent ratio, in our experience, is best achieved by adjusting the DLI probe for the maximum solvent cluster ion intensity. Gradient elution analysis should be optimized for the middle of the elution, by pressure or cluster intensity, to maintain the best response throughout the gradient. These approaches assume that the analyte behaves like benzoic acid. To test the versatility of the above tuning procedures, we analyzed a number of compounds in a 60/40acetonitrile/water mixture. Standards of 100-500 ng of benzophenone, diethyl 3,4-furandicarboxylate, trichlorophenol, diethylstilbestrol, dienestrol, o-phthalic acid, 1,2,3-benzenetricarboxylicacid, and 4-hydroxybenzoic acid all gave a maximum protonated molecular ion (moleculm ion in the case of trichlorophenol) signal at 0.280 f 0.020 torr. From Figure 5, the pressure which gives the best signal for benzoic acid was 0.275 torr. This agreement between source pressures for various compounds and the observation that solvent cluster ions generally maximize (within a 10% tolerance) along with the (M + H)+ion for the above compounds, demonstrated the usefulness of these procedures in tuning. Temperature Effects. The above experiments were carried out at source temperatures between 190 and 210 "C. A relationship was observed between fragment intensity and temperature: higher temperatures produce more fragmentation, as one would expect. The specific effects of temperature on fragmentation of and sensitivity for benzoic acid in different solvents was then determined. The effect of temperature of (M + H)+ and (M H - H20)+of benzoic acid and of one representative solvent cluster ion is shown in Figures 6 and 7, for different mixtures of acetonitrile/water and methanol/water, respectively. The (M + H)+/(M + H - HzO)+ratio can be varied by a factor of 100 by changing the temperature between 140 "C and 340 "C. It is not surprising that fragmentation increases with source temperature,
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982 l"",
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solvent clusters decreased with increasing temperature, as expected because of the positive AGO for the stepwise formation of proton-bound clusters (16, 17). The optimal operating conditions for our analyte were found to be in the broad temperature range of 200-300 "C. If sample decomposition or too much fragmentation were to occur in a particular sample, lower temperatures would probably provide an increased molecular ion intensity; there is, however, a limit. Operation of the DLI interface below 120 "C is difficult due to inefficient desolvation and vaporization. For samples where volatility is the only problem, temperatures above 300 "C should provide the greatest sensitivity. In summary, the sensitivity of the technique for the (M H)+ion of benzoic acid did not change greatly with solvent composition over the temperature range 200-300 "C. The solvents containing high percentages of water showed the least dependence on temperature. These mixtures with high water concentrations tend to produce a more intense protonated molecular ion and exhibit less fragmentation. These trends of the variation of fragmentation and sensitivity with temperature were observed for all the solvent ratios studied.
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o f benzoic acid at varlous solvent ratlos of
LITERATURE CITED (1) Baldwln, M. A.; Mclafferty, F. W. Org. Mass Spectrom. 1973, 7, 1111. (2) Jennlngs, K. R. "Gas Phase Ion Chemlstry"; Bowers, M. T., Ed.; Academlc Press: New York, 1979;Vol. 2, pp 123-151. (3) Morgan, R. P.; Hayward, E. J.; Steel G. Org. Mass Spectrom. W79, 14, 627-628. (4) Mather, R. E.; Todd, J. F. I n t . J. Mass Spectrom. ion fhys. 1979, 30, 1. (5) Maylin, G. A.; Henion, J. D. Blomed. Mass Spectrom. 1980, 7, 1 15- 121. (6) Henion, J. D. Anal. Chem. 1978,5 0 , 1687-1693. (7) Hewlett-Packard LClMS Interface Operator's Manual, p 9. (6) Fleld, F. H.; Munson, M. S. B.; Becker, D. A. "Ion Molecule Reactlons In the Gas Phase"; American Chemical Society: Washington, DC, 1966;Adv. Chem. Ser. No. 58, pp 167-192. (9) Voyksner, R. 0.; Hass, J. R.; Bursey, M. M. Anal. Left. 1982, 15,
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FI ure 7. The effect of temperature on the signal intensity of (M H) and (M H - H,O)+ of benzoic acld at various solvent ratios of
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but obtaining a more intense protonated molecular ion at high source temperatures is worth noting. The intensity of the (M + H)+ ion of benzoic acid was essentially constant between 200 "C and 300 "C. Above 300 "C, the (M H)+ion intensity increased, possibly because of increased vaporization of the acid from the condensate of the drop. The intensity of the
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1-12. (IO) Parker, C. E.; Haney, C. A,; Hass, J. R. J . Chromatogr. 1962,237, 233-248. (11) Hartman,,K. N.; Lias, S.; Ausloos, P.;Rosenstock, H. M.; Schroger, S. S.; Schmidt, C.; Martlnsen, D.; Milne, G. W. A. U . S . N T I S , f B Rep. 1979,NO. PB-299, 289. (12) Jennlngs, K. R. "Gas Phase Ion Chemlstry"; Academic Press: New York, 1979;Vol. 2, pp 124-126. (13) Arplno, P. J.; Guiochon, 0. J. Chromatogr. 1982,251,203. (14) Grlmsrud, E. P.; Kebarle, P. J. Am. Chem. SOC. 1973, 95, 7939. (15) Meot-Ner, M. J . Am. Chem. SOC. 1978, 100, 4694. (16) Hlraoka, K.; Grlmsrud, E. P.; Kebarle, P. J . Am. Chem. SOC. 1974, 96, 3359. (17) Kebarle, P.; Haynes, R. W.; Collins, J. G. J. Am. Chem. SOC. 1967, 89, 5753.
RECEIVED for review June 7, 1982. Accepted September 28, 1982.
