Anal. Chem. 1984, 56, 1229-1236 X lo4 C cm-2 for T = 0.03 s, 0.06 s, and 0.12 s, respectively. (This gives a diffusion coefficient of 1.26 X cm2 s-l for tert-butyl p-toluate). This indicates that the process responsible for the second electron observed in the coulometry experimentsis negligible in the potential step experiment. The discrepancies between the two experiments are probably due to different time scales and initial ester concentrations: 50 mM and 2 h for coulometry vs. 1.05 mM and 0.12 s for the longest potential step experiment. A better indication of the validity of an assumed mechanism is obtained by analyzing numerous current ratio points from each experiment. Using parameter values similar to those found assuming an EC mechanism, ECE DISPl data files were simulated and then analyzed by the EC nonlinear least-squares method. As shown in Figure 3, the resulting "best fit" did not match the simulated data. Even worse matches were obtained for simulated ECE data. Registry No. tert-Butyl p-toluate, 13756-42-8.
LITERATURE CITED (1) Schwarz, W. M.: S h a h I. J. Phys. Chem. 1965, 69, 30. (2) Marcoux, L.; O'Brlen, T. J. P. J. Phys. Chem. 1972, 7 6 , 1666.
1229
(3) Cheng, H. Y.; McCreery, R. L. J. Elecfroanal. Chem. 1977, 85, 361. (4) Cheng, H. Y.; McCreery, R. L. Anal. Chem. 1978, 5 0 , 645. (5) Chrlstle, J. H.; Osteryoung, R. A.; Anson, F. C. J . Nectroanal. Chem. 1967, 13, 236. ( 6 ) Chllds, W. V.; Maloy, J. T.; Keszthelyi, C. P.; Bard, A. J. J. Elecfrochem. SOC. 1971, 118, 874. (7) Vlslnskl, B. M.; Dryhurst, G. J. Elecfroanal. Chem. 1976, 7 0 , 199. (8) Hanafey, M. K.; Scott, R. L.; Ridgway, T. H.; Rellley, C. N. A n d . Chem. 1976, 5 0 , 116. (9) MacDonald, D. D. "Transient Techniques in Electrochemlstry"; Plenum Press: New York, 1977: Chapter 4. (10) Beilamy, A. J. Anal. Chem. 1980, 52, 607. (11) Woodard, F. E. Ph.D. Dissertation, University of North Carollna at Chapel Hill, 1982. (12) Woodard, F. E.; Woodward, W. S.; Rellley, C. N. Anal. Chem. 1981, 53, 1251A. (13) SavBant, J. M.; Tessler, D. J. Electroanal. Chem. 1975, 65, 57. (14) Feldberg, S. I n "Electroanalytical Chemistry"; Bard, A. J., Ed.; Marcel Dekker: New York, 1969 Vol. 3. (15) Hamming, R. W. "Numerical Methods for Scientlsts and Engineers"; McGraw-HIII: New York, 1962; pp 34 and 389. (16) Wagenknecht, J. H.; Goodin, R. D.; Kinlen, P. J.; Woodard, F. E. J. Electrochem. SOC., In press. (17) Hawley, M. D.; Feldberg, S. W. J. Phys. Chem. 1966, 70, 3459. (18) Amatore, C.; SavQant, J. M. J. Electroanal. Chem. 1977, 85, 27.
RECEIVED for review September 26,1983. Accepted February 21, 1984.
Gradient Liquid Chromatography/Mass Spectrometry Using Microbore Columns and a Moving Belt Interface M. J. Hayes,' H. E. Schwartz, Paul Vouros,* and B. L. Karger* Barnett Institute of Chemical Analysis and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115
A. D. Thruston, Jr., a n d J. M. McGuire Athens Environmental Research Laboratory, US. Environmental Protection Agency, Athens, Georgia 30613
Hlghperfonnance gradlent microbore LC/MS Is demonstrated by use of spray deposition onto a moving belt surface. A slmpllfled nebulizer deslgn Is shown to provide convenlent operatlon and high transfer efflciency over a wide range of flow and solvent conditions. I t Is further shown that the extracolumn varlance of the LC/MS system is sufflclently small to allow the use of relatively short 5 pm and 7.5 pm reversed-phase microbore columns wlthout signlflcant loss in resolutlon. Relatlvely hlgh mlcrobore flow rates (-100 pL/mln) are posslble for fast analysls and sensltlvlly. The LC/MS technlque Is applied to the analysis of phenols In an aqueous coal gasiflcatlon sample. Detection llmlts for full scan LWEI-MS are found to be - 6 ng for some representative phenols.
In recent years, increasing interest has been shown in on-line liquid chromatography/mass spectrometry (LC/MS) for analysis of polar and/or nonvolatile compounds. Two major interface approaches have been developed based on direct liquid introduction (1) and moving belt (2) principles. At present, direct liquid introduction, including the promising thermospray technique ( 3 ) )has been shown to yield useful Current address: Drug Metabolism Subdivision, Ciba-Geigy Corp., Ardsley, NY 10502. 0003-2700/84/0356-1229$01.50/0
spectra of nonvolatile and thermally labile compounds. The moving belt interface is also a promising technique, allowing the selection of the ionization mode of choice, along with the potential for employing a variety of surface ionization techniques for the analysis of substances which are difficult to vaporize (4,5). Recently, we published an extensive study on the use of spray deposition, first reported by Smith and Johnson (6))for the optimization of the chromatographic performance of the moving belt system (7). It was shown that it is possible to perform on-line LC/MS with normal bore columns (4.6 mm Ld.), using either normal or reversed-phaseLC. Extracolumn contributions to band broadening were sufficiently reduced to maintain chromatographic integrity of the peaks eluting from the column. Some difficulties remained, however, in the handling of mobile phases of high water content at the flow rates commonly used for normal bore columns (- 1mL/min). It was necessary to use reduced flow rates at the expense of analysis time and sensitivity or to split the effluent, again reducing sensitivity. Microbore columns have been utilized to overcome the flow rate problem with moving belt systems. The reduced flow rates required for rapid mobile phase velocities significantly decrease the problem of solvent removal. For example, Games et al. (8,9) have shown that LC eluents containing high water content can be successfully flowed onto the moving belt, although such eluents may require the addition of a miscible cosolvent, e.g., ethanol, to the belt. Microbore LC/MS has 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
also been demonstrated with a vacuum nebulizing interface (10) and with direct liquid introduction (11). In this work we have extended the moving belt spray deposition approach for use with microbore columns in order to improve our previous system (7). The nebulizer has been redesigned in order to accommodate the low flow rates and to minimize band broadening contributions from the interface, It is shown that high efficiency 1mm microbore columns can be used without appreciable loss in resolution. Additionally, it is shown that full range gradients, using a previously developed chromatographic system (12),can be performed at microbore flow rates without having to alter the nebulization conditions during the coufse of the gradient run. Nebulization thus provides convenient optimization of deposition conditions to suit the chromatographic system. As an application of this approach, we have examined the LC/MS analysis of phenolic compounds in aqueous samples originating from coal gasification processes. Several compounds in the sample were identified from retention and mass spectral data. Their presence was confirmed by using various techniques, including normal bore LC/MS. EXPERIMENTAL SECTION Mass Spectrometer and Interface. A Finnigan 4000 mass spectrometer (San Jose, CA) equipped with a moving belt LC/MS interface with Kapton belts was used in all the microbore experiments. Electron impact (EI) mass spectrometry was performed with an indicated source temperature of 250 "C. Chemical ionization (CI) was conducted with the ion source at 160 "C and with methane as the reagent gas at a pressure of 0.30 torr. The belt speed was held constant at 2.5 cm/s, and the vaporizer heater was set to 220 "C. The infrared heater in the moving belt housing was used for all experiments. Normal bore HPLC/MS analysis of the coal gasification water sample was also conducted with a Finnigan 3200 equipped with a similar moving belt interface and a nebulizer, as described earlier (7). The indicated source temperature was 60 "C and that of the vaporizer 165 "C. Spray deposition was employed for transfer of the chromatographic effluent to the belt surface. For microbore HPLC/MS, our previously developed nebulizer was modified by the addition of a new transfer line assembly and spray tip; see Figure 1. The transfer line (A, in Figure l),made from a 19 cm long by 0.009 in. o.d., 0.0045 in. i.d. stainlesssteel tube (Hamilton, Reno, NV), was connected to the outlet of the LC column via a capillary column butt connector (Supelco, Inc., Bellefonte, PA). The connector was modified by drilling out the ferrule to '/I6 in. for half of its length and inserting a '/I6 in. o.d., 0.10 in. i.d. tube. The 0.009 in. 0.d. transfer line was then inserted, as shown in the inset of Figure 1. This arrangement resulted in a zero dead volume connection between the column and the transfer line. The transfer line was attached to the body of the nebulizer (B in Figure 1)with a standard tube fitting and a graphite capillary ferrule. The gas orifice was made by fusing two pieces of glass together, one of 1.5 mm o.d., 0.75 mm i.d. tubing (E in Figure 1) and the other, which served simply as a holder, of 1 / 4 in. o.d., l/s in. id., glass tubing (H in Figure 1). The parallel tubes in the spray orifice allowed the gas flow rate and the relative positions of the two tubes forming the orifice to be optimized independent of one another. Other details of the construction and operation of the nebulizer are similar to those outlined in ref 7. Chromatographic System. The gradient system and the dynamic mixer were as previously described (12). Two Waters Associates (Milford,MA) Model 6000 A pumps, in which the pulse dampeners were bypassed, were driven by a Waters Model M660 gradient controller. Two flow rate converters (Waters Associates) were installed between the gradient controller and the two pumps to provide suitable flow rates in the 40-400 pL/min range. Either an air actuated Valco Model CV-6-UHPa-N6Osix-port valve or an electrically actuated, Valco Model ACF 4U.2 (0.2 p1) valve was used for injection. With the latter valve, a moving injection technique (13) was employed in order to minimize extracolumn dispersion in the injection valve. Two reversed-phase stainless steel microbore columns were packed in our laboratory. One column, 350 X 1.0 mm i.d., con-
inset 2 (not t o s c a l e )
E F
\
Flgure 1. Diagram of spray deposition device: (A) stainless steel (SS) transfer line from LC; (B) SS body of spray device: (C) retaining cap; (D) O-ring seal; (E) Pyrex holder for nebulizing gas orifice; (F) Kapton moving belt; (G) gas inlet; (H) Pyrex tip to form nebulizing gas orifice. Inset shows the capillary butt connector. See text for details.
tained 7.5-pm Zorbax-BP-ODS (Du Pont, Wilmington, DE) and the other, 25 X 0.1 cm, was packed with 5-pm Supelcosil C-18 (SupelcoInc., Bellefonte,PA). The columns were fitted with Valco Model CEF-1 (0.01 in. hole) micro end fittings in which 2-pm frits were inserted (Mott Metallurgical, Farmington, CT). The microbore columns were connected to the injection valve and UV detector cell with 40 mm by 0.007 in. i.d. connecting tubing. The LC/MS interface was connected directly to the outlet of the microbore column, as described above. Normal bore HPLC was conducted with a commercial 250 X 4.6 mm i.d. Supelcosil LC-8 column. A Kratos Model 773 variable wavelength detector, in which special microbore flow cells (0.5 pL, 1 mm path length) were installed, was used for experiments in which UV detection was employed. The wavelength was set at 280 nm. Chromatograms were recorded on a 10-mV strip chart recorder (Linear Instruments, Irvine, CA). Chemicals. Acetonitrile, water, and methanol (Baker Analyzed Reagents, Doe and Ingalb Inc., Medford, MA) were HPLC grade. Trifluoroacetic acid (TFA) was purchased from Pierce Chemical (Rockford, IL). Acetic acid was purchased from Burdick & Jackson. The phenol standards and uracil were obtained from Aldrich Chemical (Milwaukee, WI) and deuterated catechol (catechol-d4)was synthesized, as described previously (14). Aqueous environmental samples were obtained from the effluent of experimental gasification processes through the courtesy of the Pittsburgh Energy Technology Center of the U.S.Department of Energy. The samples were filtered through a 0.45-pm membrane filter and a Fiberglas prefilter by means of a sample filtration kit (Rainin, Woburn, MA). An aliquot of the filtrate was injected directly into the LC column without further pretreatment. Mobile phases were prepared by premeasuring the components in the volumetric proportions specified in the text prior to mixing. Degassing over a 15-min period was carried out by applying vacuum while stirring the mixture. RESULTS AND DISCUSSION Nebulizer Design. Initially, we attempted to use our original design (7) with microbore columns but found that this design caused significant band broadening at microbore flow
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
rates (30-100 pL/min), apparently due to the dimensions of the spray orifice. We therefore constructed a new, smaller tip, as described in the Experimental Section and Figure 1. With an orifice size of 0.75 mm, the use of mobile phase flow rates of up to 300 pL/min at 100% water was possible. Beyond this flow rate, a stable base line could not be maintained. Several smaller orifice sizes were also evaluated (0.25 mm and 0.35 mm), but these were unable to handle flow rates of more than 40 pL/min. The reduced dimensions of the redesigned nebulizer provided for less solvent holdup a t the tip compared to the previous design, thereby reducing band broadening (see below). Moreover, the parallel arrangement of tubes A and H in Figure 1allowed for optimization of the relative positions of these tubes independent of one another. Extracolumn Variance. It was important to determine quantitatively the extracolumn variance due to the interface. Previously,Games et al. (15) found low variances by using the direct deposition approach at 10pL/min with 100% methanol as the mobile phase. These authors observed that with mobile phases of high water content, however, addition of ethanol onto the belt surface was necessary in order to obtain useful LC/MS chromatograms. The variance of the LC/MS interface using the nebulizer of Figure 1 was measured by connecting the inlet tubing of the interface directly to the 0.2-pL Valco valve, as previously described by Lauer and Rozing (16). The resulting C? is then the sum of the variances of the injection valve (u2""valve) and interface ( UPint)
(1) + g'int It was found by Scott and Simpson (17)that the variance contribution of an injection valve similar to that used in this work is quite small (0.05 pL2 at a flow rate of 20 pL/min). Therefore, ( T can ~ be ~ assumed ~ to equal uzint. In our previous report (7)we determined the variances of tailed Gaussian peaks by means of a computer program which calculated the central moments of the peaks. Recently, Foley and Dorsey reported (18)that such peaks (i.e., exponentially tailed Gaussians) could be accurately characterized by empirical equations in terms of easily measured parameters. The equations for calculating the number of theoretical plates, N , and the second central moment (variance, 2)were reported to give good accuracy for peak asymmetry values 12.76. We have confirmed these results by comparing this manual method with our previously employed computer method for variance calculations. Since the precision of the variances was found to be better for the manual method, we decided to use this approach for calculation of N and u2in the present work. We determined initially the variance contributed by the interface, u2int,as a function of the mobile phase flow rate. In this experiment, the flow rate was varied from 20 to 200 pL/min and the variance resulting from a 0.2-pL injection of a uracil solution was determined. The nebulization conditions were optimized for each flow rate. In addition, the mass spectrometer was operated in the single ion mode at a Sampling rate of 0.5 s/scan. A linear plot was obtained between g2int and flow rate. By use of the Taylor dispersion theory of open tubes (191,it can be calculated that 35% of the measured u2int is due to diffusion effects in the transfer line. The remainder of & appears to be caused by the spray deposition process itself. From these results a value of gPint= 0.7 pL2 was measured at a flow rate of 20 pL/min, the optimum flow rate for microbore columns of this type. Using the procedure of Reese and Scott (20),we have also measured the combined variance contribution from a UV detector (equipped with a 0.5-pL flow cell) and a submicroliter injection valve and obtained a value for the same flow rate of 1.5 pL2, twice that of the LC/MS interface. ~'sys
=
I
1231
A I
B
g'valve
4
8 RE-Eh-lOh
16
2 IM N
2c
J
Comparison of UV and MS chromatograms using a 5-pm microbore column: column, 250 X 1.0 mm, Supelcosil C18, 5 pm; solutes, resorcinol, 1,5dihydroxynaphthol,and 2-methylphenol (200 ng of each): flow rate, 40 mL/min; mobile phase, 41 % acetonitrile, 59% water, 0.1% TFA added; (A) LCNV at 280 nm 0.015 AUFS, (9) LCIMS, selected ion chromatograms. Figure 2.
The measured values of the extracolumn variance can be used to calculate the expected loss in resolution for typical high-performance microbore LC/MS systems. The percent loss in resolution, M , can be expressed as
where ucol= Vo(l + k?/Nl12 and Vo is the void volume in the column. We have tabulated the losses in resolution as a function of k'for columns of 50000 plates/m and 60000 plateslm, found for the 7.5-pm and 5-pm HPLC microbore columns, respectively. It can be seen that the losses in resolution due to the LC/MS are quite small and are in fact considerablyless than those calculated for the LC/UV system, which was specifically designed for microbore work. These data reveal that the moving belt system is well suited for use with microbore columns. With this favorable result we explored the use of the 5-pm column with the on-line LC/MS system. In Figure 2 the UV and MS chromatograms are shown for these substances (resorcinol, 1,5-dihydroxynaphthol, and 2-methylphenol). Selected ion plots are shown for the MS chromatograms, since TFA in the mobile phase obscures most peaks in the total ion plots. In the case of the UV detector, the column was connected to the flow cell via the shortest possible connecting tubing (4cm X 0.007 in. i.d.). Although visually the chromatograms appear the same, measurements show significantly higher plate counts for the MS system for the first eluting peak (11200 for MS, 8200 for UV). For this early eluting peak, the extracolumn effects from the UV detector are most prominent.
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
Table I. Percent Loss in Resolution for Two Microbore Column Systems SYSTEM I(7.5 pm) k’
AR,,,%
0
2
11.9 3.4 1.6
3
0.9
4
0.6
1
/
system I
SYSTEM I1 (5 pm)
ARuv,% ARM,,%
21.3 7.0
3.3 1.9 1.2
20.2 6.4 3.0
12.6 6.1
1.7 1.1
3.6 2.3
20
40
60
80
100
Z ACETONITRILE IN WATER
Figure 3. Plot of solute transfer efficiency to the belt vs. mobile phase composition. Flow rate was 40 pLlmin.
Previously, with normal bore systems (8)we found a 40% loss in resolution for an unretained peak during HPLC/MS with the moving belt. Thus, the use of the redesigned nebulizer with microbore columns results in significantly improved performance. Traqsfer Efficiency. In our previous work (3,it was found that it was not possible to perform a reversed-phase gradient over a wide composition range without altering the nebulizing conditions to compensate for the changes in solvent volatility. Since significantly lower flow rates are used with microbore columns, we decided to examine whether the new system could be used over a wide gradient range under constant nebulizing conditions. Test peaks were generated by injecting 1pL of a uracil solution (50 ng) into a Valco six-port valve connected to the MS with a capillary tube of 10-pL volume. The nebulizer was operated at a constant gas pressure of 5 psi with the gas heated to 40 “C. The mobile phases were varied between 0 and 100% acetonitrile a t a flow rate of 40 pL/min. The transfer efficiency was measured by comparing the area obtained by flowing the mobile phase directly onto the belt to that found by spray deposition. As seen in Figure 3, except for the losses with mobile phases containing a very high water content (Le., 190%),the transfer efficiency is close to 100%. The error in the measurement of transfer efficiency is estimated to be 5-lo%, particularly at the high water content values, due to difficulties in flowing the solvent directly onto the belt. It may also be noted that the variance of the test peaks did not change as the mobile phase composition was varied. The results of Figure 3 indicate that wide range gradient chromatograms can be generated at 40 pL/min without having to alter the nebulizer conditions during the course of the gradient run. In cases where solutes are eluting at very high water content under isocratic conditions, it is, of course, possible to adjust the spray conditions to optimize the transfer efficiency. Above -80 pL/min, we were not able to run full gradients without having to make some minor adjustment in the spray pressure during the course of the run. When these adjustments are made, it is possible to run full gradients at flow rates of at least 100 pL/min and obtain low extracolumn variances and stable base lines. Influence of Flow Rate on Sensitivity and Efficiency. Detector sensitivity, important in LC/MS analysis, is closely related to the transfer efficiency to the belt, assuming all other variables are optimized for a given analysis (e.g., desorption temperature, etc.). The mass spectrometer is a mass flow sensitive detector and the peak height should thus increase in direct proportion to the flow rate of the eluent, while peak area should remain constant. In order to determine the relative sensitivity for the LC/MS system, defined as the product of area and flow rate at constant solute mass, the following experiment was carried out:
33.9
column 35 X 0.1 cm N = 17 500 (50 000 plates/m)
V, = 206 pL ( E 0
AR,,,%
=
0.75)
system I1
column 25 x 0.1 cm N = 1 5 000 (60 000 plates/m) V,,= 147 pL ( E = 0.75)
for both systems
F = 20 uLlmin u2int(MS)’=0.7 pLz
1 pL of a resorcinol solution (400 ng) was injected onto the 7.5-pm column, with a mobile phase consisting of 41 % acetonitrile, 59% water and 0.1% TFA. Nebulization conditions were optimized for each flow rate. It was found that over a large flow rate range, Le., from 20 pL/min to 250 pL/min (typical microbore range), the relative MS sensitivity was, indeed, a linear function of the flow rate, consistent with a mass flow sensitive detector. Flow rates higher than 250 pL/min were not considered as, in this case, it would be difficult to evaporate all the solvent to obtain a stable base line. Because of the low flow rates of the microbore column, the interface can be conveniently operated well above the optimum flow rate of the column. It was found that a 1.9-fold gain in relative sensitivity can be obtained by increasing the flow rate from 40 to 100 pL/min. Hence, for a given column, depending on the separation, one should decide whether to use a relatively high flow rate with the advantages of higher sensitivity and faster analysis or a relatively low flow rate resulting in optimum resolution. A comparable increase in flow rate with normal bore columns would be difficult to handle due to the relatively large flow rates, especially at high water contents. It should also be mentioned that the mass capacity of microbore columns matches well that of the MS. With typical microbore systems, 1pg per component represents a reasonable upper limit for the mass capacity of the column. When more than 1 pg of component is injected into the MS, contamination of the ion source may also occur rapidly. Comparing conventional columns with microbore columns and injecting the same mass on each column, there would, in principle, be no gain in sensitivity with a microbore system, since the inherent lower flow rates of the latter cancel out any gain achieved by the reduced solute dilution of a microbore system. In practice, however, one can benefit in terms of detection limits with the low flow rates from microbore columns, due to the reduced solvent background noise obtained. This effect has also been seen by others ( 4 ) . The background noise caused by solvent ions can vary considerably in LC/MS, as the nature of the mobile phase, including additives (e.g., TFA), is altered. There are two basic mechanisms that contribute to the solvent noise effects. The first is direct interference from the solvent that occurs below about 150 amu, due to solvent vapor that leaks into the mass spectrometer through the vacuum locks. The second source of noise is particularly noticeable with the use of mobile phases of high water content and is manifested by the appearance of weak background signals at almost any mass over a wide
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
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SOLUTES A RCSORCINOL
x CATECHOL 0
200
400
600
1.5-DIOH-NAP
800
CONCENTRATION INJECTED (ng/pL)
Figure 4. Calibration curves for resorcinol, catechol, and I ,5-dihydroxynaphthol: internal standard, catechol-d,; column, 350 X 1.O mm Zorbax-BP-ODs, 7.5 pm; solvent A, 5% acetonitrile, 95% water, 0.1% TFA; solvent B, 95% acetonitrile, 5 % water, 0.1% TFA; gra70% B in 40 min; flow rate, 100 hL/min. dient, 5
-
(-200 amu) mass range. Although the origin of this noise is not totally clear, it is reasonable that the vaporization of excess solvent entering the ion source causes the release of residual materials which collect on the belt surface over the course of time. For example, in the transfer efficiency experiment of Figure 3, the base line noise increased for mobile phases containing very high water content. Detection Limits. We next generated calibration curves and determined the detection limits for a series of low molecular weight phenolic standards. The MS was operated in the full scan mode from 45 to 250 amu, and the detection limit was defined as the mass of solute injected onto the column that produced a signal to noise ratio of 3 for the molecular ion of each compound. The chromatographic conditions were chosen such that they would be identical with those necessary for an actual sample containing phenols (to be discussed in the next section). Catechol-d, (100 ng/pL) was selected as internal standard since it elutes at an intermediate k’value, is chemically similar to the solutes of interest, and does not interfere with the analysis. Figure 4 shows calibration curves for resorcinol, catechol, and 1,5-dihydroxynaphthol. Under the given conditions, the detection limit for the three compounds was approximately 6 ng. The calibration curves were linear (r 2 0.97) up to 750 ng injected on the column which was near the mass loading limit for the column. The low nanogram detection limit is significant, since it is based on a full scan spectrum. In addition, Figure 4 represents a particularly difficult example, since for the molecular weight range (1000, is still a difficult challenge for most of the exisiting LC/MS instruments (3).
One approach for increasing the volatility of peptides has been to block terminal amino and carboxylic groups, and in some cases also the ionic and polar side chains, by chemical derivatization (46).This procedure has been successful for low molecular weight peptides but still has not produced large peptides volatile enough to be subject to electron impact (EI) or chemical ionization (CI) mass spectrometry. Characterization of nonderivatized peptides by mass spectrometry has been performed efficiently by field desorption (FD) by 252Cfplasma desorption (252Cf-PD)(8), and in particular by fast atom bombardment (FAB) (9, 10). The first two methods require off-line sample preparation, incompatible with direct coupling to LC, and the latter has recently been proposed as an on-line detector of LC eluants using the moving belt system (11). The combination with chromatographic separation adds an important dimension to the analysis of impure samples and samples in biological matrices and, in particular, for the detection of oligopeptide mixtures resulting from enzymatic digestion used in peptide mapping. Developments in the methodology of LC separation of peptides and proteins have been reviewed extensively
(a,
(12-14).
Thermospray, defined as the complete or partial vaporization of a liquid stream by heating as it flows through a capillary, has recently been demonstrated as a versatile interface for combined LC/MS (15-18). The heat is supplied electrically and controlled by a feedback system to maintain a constant level of vaporization. The optimal temperature
0003-2700/84/0356-1236$01.50/00 1984 American Chemical Society
