Continuous Mass Spectrometric Monitoring of a Liquid Chromatograph with Subnanogram Sensitivity Using an On-Line Computer F. W. McLafferty,’ Ruedi Knutti, Rengachari Venkataraghavan, P. J. Arpino, and B. G. Dawkins Department of Chemistry, Cornell University, nhaca, N. Y. 14853
A llquid chromatograph (LC) for which eluted sample components are detected by continuous direct introduction of the solutions into a chemical ionization mass spectrometer (CIMS) has been coupled to a laboratory minicomputer (COM). The resulting LC-MS-COM system shows many advantages now well established for GC-MS-COM systems, such as the real time preparatlon of reconstructed liquid chromatograms, mass chromatograms, and multiple ion detection. Detection specificities made possible by the lndividual mass peaks are superior, and the subnanogram detection sensitivities achieved are at least comparable to those of any other detector, and are applicable to most samples meeting the low vapor pressure requirements of direct chemical ionization.
Liquid chromatography (LC) is experiencing an explosive growth in analytical applications reminiscent of that shown by gas chromatography a decade or more ago ( I ) . One of the most serious instrumental problems limiting further applications is the availability of detector systems of suitable sensitivity and specificity ( 2 ) . The ultraviolet spectrometer is the most generally used detector of high sensitivity, but its application is limited by the UV absorptivity of the sample. For example, the lower limit for detection of 2-naphthacyl derivatives of fatty acid esters (log 6254 n m = 4.1) was found to be 4 X g (3) and, even with multiple wavelength information, the specificity of UV detection is relatively poor. A variety of alternative detectors of comparable sensitivity and broader applicability have been proposed recently (4-8). We have described a system (5, 6) in which -1% of the LC effluent solution is introduced directly into the ion source of a chemical ionization mass spectrometer (CIMS; the solvent acts as the ionizing reagent) to provide specific as well as sensitive detection of almost any eluted component having sufficient vapor pressure. The “direct chemical ionization” (9) effective in this system makes it possible to detect compounds of much lower vapor pressure (molecular weights >>lo00 for nonpolar compounds) than other ionization techniques in which the sample is vaporized a t atmospheric pressure (7, 8).
We describe here the combination of such an LC-CIMS with an on-line minicomputer. The well-known capabilities of gas chromatography-MS-computer (GC-MS-COM) systems (10) make this a logical extension of the LC-CIMS technique. For GC detection, the mass spectrum provides much more information concerning the eluting component than does a conventional single-parameter detector. Using an on-line computer for real-time data acquisition and reduction makes it possible to obtain a complete mass spectrum of the effluent every few seconds and to display the GC results as a series of “mass chromatograms” ( 1 1 ) .Continuous computer monitoring of a single or a few mass T o whom correspondence should be addressed.
peaks gives greatly increased sensitivity as well as selectivity. However, basic differences in LC-MS and GC-MS lead to contrasting requirements in particular areas. In most GC-MS systems, the bulk of the carrier gas is removed by a separator before the effluent enters the MS; in this LC-MS system (5, 6), a 1:lOO splitter must be employed, increasing the sensitivity requirements for the MS detector system. The common GC carrier gases are of such low molecular weight that they do not interfere with the mass spectrum; the LC solvents commonly employed are of higher molecular weight, and telomerization in the CI source can give solvent peaks above mle 100. Because the most common LC detector used for trace analysis (the UV spectrometer) has high sensitivity only for particular classes of compounds, it is especially important in the LC-MS system to be able to distinguish solute and solvent peaks for most solute-solvent combinations. A special objective of this work was to see if computer techniques could decrease the LC-MS detection threshhold to the subnanogram range to make it clearly comparable or superior to the UV detector for any sample of sufficient volatility. EXPERIMENTAL The LC-MS system, which has been described (5, 61, uses a Waters ALC 202 LC; a reverse-phase c-18chemically bonded column was used in the examples described below. This was attached to a Hitachi RMH-2 MS (5400-V ion acceleration, 500-eV ionizing electrons) through a capillary-splitter interface which introduces -10 pl/min of the LC effluent continuously into the CI source of the MS. With a liquid nitrogen trap directly above the source acting as a cryogenic pump, source housing pressures were typically Torr. The system design leads to a very high pressure gradient in the ion source, so that the source pressure is adjusted to give reasonably low amounts of the telomerized solvent ions. Computer Hardware. The MS output from a Bendix 4700 continuous-dynode electron multiplier is sent to a Keithley 427 current amplifier; this is connected through a Preston GMAD-2 analog-to-digital converter (ADC) to a Digital Equipment Corporation (DEC) PDP-11/45 computer. The computer programs for this operation use 12K of core memory and a 1.2M word disc. Data display is accomplished with a DEC-GT/40 cathode ray tube system; data exchange between the GT/40 and 11/45 is accomplished through a bus window. A programmable 1-MHz clock with a 32-bit elapsed time counter serves as an accurate time base. The interface contains hardware for peak detection; a peak starts when a datum exceeds a programmable threshhold and ends when three (or a selected number of) contiguous data points are collected below this threshhold. The interface interrupts the central processing unit (CPU) of the computer only a t the end of a peak to transfer the data points, a flag word, and a 32-bit time value. The ion accelerating voltage can be changed by computer command through a digital-to-analog converter. The interface also contains a computer driven relay to initiate magnet scans. Software. Programs are written in assembler language (MACRO11), and listings are available on request from the authors. The display oriented LC-MS-COM system can be operated in two modes: LCMS for continuous, cyclic mass scans, and MID for multiple ion detection. In LCMS, the computer directs sequential acquisition of mass spectra of the LC effluent by cyclic magnetic scanning. Keyboard adjustable parameters include downscan time, flyback time, scan rate, delay after scan start before commencement of data acquisition (to allow magnet stabilization), and sampling interval. ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
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500r
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5 IO 15 20 Time After I n j e c t i o n , minutes
Flgure 1. Mass chromatograms for m/e 189, 203, 215, and the summed ions of m/e 160-550, in arbitrary units of ion current; LC g of trilaurin, 4% of effluent to MS, signal separation of 50 X threshold 100 units
lo-'
For the real-time data processing, the computer checks to see that the data points for a particular peak meet a minimum width requirement and then calculates the peak height, area, and centroid time. These data are stored on disc and also transferred to the GTl40 for immediate display of peak height vs. scaled time values; mle markers calculated from stored m a w t i m e values are displayed on the abscissa. The current spectrum is generated on the bottom of the screen with the preceding two spectra above it for comparison. Time-to-mass conversion is done using data from a separate mass calibration run (perfluorokerosene) employing a third-order polynomial and logarithmic time values. Off-line data analysis programs, including those for the display of reconstructed liquid chromatograms (Figure 1) and mass chromatograms ( I I ) , are similar to those described for GC-MS-COM systems, with CRT, plotter, or line printer output. The MID mode is similar to ones previously described for GCMS-COM systems (11).A single peak can be monitored by setting the MS a t the corresponding magnetic field, or neighboring peaks can be measured sequentially by computer-directed changes in the ion accelerating potential through the DlA hardware. Data points are typically collected a t a rate of 50 KHz; the computer sums these values for a predesignated time interval (for example, 20 msec) and stores the average of the collected sum. The values for the abundance of a particular peak as a function of time are displayed in real time on the GT/40 to yield a mass chromatogram (Figure 2); the running sum of the ion current values is also displayed to give the integral of the chromatogram. Further time averaging and base-line subtraction can also be carried out on the displayed chromatograms by the MID software. In another program, the ion accelerating voltage is stepped to measure alternately the peak and its adjacent base line, with the difference in ion current used as the peak height measurement; however, in the cases tried, this did not give any particular advantages in signal/ noise in comparison with normal single ion monitoring.
RESULTS AND DISCUSSION LCMS Mode O p e r a t i o n . For the ideal chromatographic detector, all useful mobile phases (but no samples) should be "transparent". For GC-MS, the carrier gases are generally of such low molecular weight (MW) that they cause no interference. Most useful LC solvents are of MW < 125; even those which tend to telomerize extensively in the CI 1504
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
Integrated
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I p i pure methanol
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0
--I
I
I
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5 IO 15 20 Time After I n j e c t i o n , minutes
Figure 2. Single ion detection of m/e 215 for LC separation of trilaurin, 4YO of effluent to MS. "Visicorder" is an analog recording; "4 sec averaging" is computer summing of 50-kHz ion signals for 4-sec intervals: "integrated" is the running sum of these signals
source, such as H20, CHBOH,and CH&N, give almost no peaks of m / e > 150 in their mass spectra at the operating pressures employed- in this study. Fortunately, most samples for which LC is more appropriate than GC give the most abundant peaks in their CI spectra above m/e 150, so that almost all eluted sample components with sufficient vapor pressure for direct chemical ionization (12) are detectable by MS-COM. For study of an unknown mixture using the LCMS mode, the MS scan time is set to be substantially less than the expected widths of the LC peaks (e.g., a scan rate of 10 sec/decade), and mass spectra are collected repetitively during the LC run. The simplest method of LC peak detection is to have the computer display a reconstructed chromatogram of the total ion signal for all peaks of masses above those from the solvent ( m / e > 160 in Figure 1). For cases in which the CI mass spectral behavior of the solvent ( 1 3 ) has not been studied or is not easily predictable, such as in gradient elution, peak detection by visual inspection of the CRT-displayed spectra, either during or after the run, may be desirable. In our use to date, it has not been necessary to use solute peaks of low mass, although these are often easily discernible in the side-by-side CRT inspection of mass spectra of the effluent, or they can be displayed directly by computer subtraction of the CI-MS spectrum of the solvent measured from the LC base line adjacent in the chromatogram.
T o illustrate the application of the LCMS mode, 50 ng of a sample of trilaurin, MW 638, was injected a t the head of the LC column, eluted with methanol, and mass spectra were scanned repetitively (20 sec, m/e 70-700) on the -1% of the effluent directed to the MS. The CI spectrum of trilaurin with CH30H as the ionizing reagent shows a base peak at m/e 215, in contrast to the 200' isobutane CI spectrum (14), as well as an order-of-magnitude smaller (M + H ) + peak a t m/e 639; the m/e 215 peak presumably is protonated methyl laurate formed by reaction with the solvent ions. A reconstructed liquid chromatogram (Figure 1) of masses 160-550 indicates a number of eluted components from the supposedly pure sample. A mass chromatogram using m/e 215 clearly shows the trilaurin eluting with a retention time of 17 minutes. The mle 215 peak eluting a t -9 minutes was reproducible, however. This is probably from lauric acid present in the sample as an impurity, although it is possible that some sample solvolysis occurred in the inlet system. The largest peak in the reconstructed chromatogram arose chiefly from mle 189 and 203 ions (Figure 1).These were found even with the injection of pure solvent, and their variability in height with mode of injection indicated that they arise from impurities introduced from the septum. Many of the smaller peaks in the reconstructed chromatogram also appear to be impurities from the inlet system, and scrupulous cleaning is necessary to achieve low noise chromatograms because of the generally high sensitivity of CIMS to all compounds of sufficient volatility. However (Figure l), greatly improved noise levels are shown by the individual mass chromatograms; the specificity of these is generally much higher than chromatograms from other detectors, even the multiple wavelength UV spectrometer. Note that if the CIMS information is not sufficient for identification, the eluted sample components indicated in the mass chromatogram can be collected with relative ease because only -1% has been used for CIMS detection, and because of the relatively large solvent volumes involved. Under normal operating conditions, collection of 0.1-ml samples of eluate solutions will give several samples across a single LC peak, and evaporation of these in a sample holder for normal direct probe MS introduction provides sufficient sample for identification from the electron ionization (EI) mass spectrum at the nanogram level. Structural Information on Eluted Components. A well-known advantage of electron-ionization MS mohitoring of GC separations is that the mass spectral information can provide a t least partial structural characterization of the separated compounds. CIMS is especially useful for molecular weight determination, although it is of more limited applicability for structure elucidation (13, 14). A particularly favorable case, which also takes advantage of the low vapor pressure requirements of direct CI, is the amino acid sequencing of oligopeptides; complete sequence information can be obtained from many underivatized tetraand even pentapeptides (9). In attempting to develop a method for polypeptide sequencing ( 1 5 ) through degradation to oligopeptides which are separated and analyzed by
LC-MS, we have been able to obtain complete sequence information from the injection on column of as little as g of derivatized oligopeptides. Despite the fact that much less sample would be required if the 1OO:l split were not necessary, this
