Finally, the validity of employing “half-intensity” integrations is tested. When full resolution of a profile is lacking but the central portion of the profile is not affected by interferences which occur on only one side of the profile, then the profile is scanned from its free side and the intensity integration is performed only for the free half of the profile, that is, u p to the peak-height channel. A user option programs profiles for half-intensity integration a t the time of acquisition. The results in Table V indicate excellent precision for 9Be, a n isotope for which all lines were computed by half -intensity integration. Also, the effect of mixing half-intensity and full-intensity calculations
has not harmed the precision for integrated intensity us. peak-height intensity for those isotopes so indicated in Table V .
ACKNOWLEDGMENT The authors are indebted t o M. D. Lawless for designing and building parts of the acquisition system. Received for review June 12, 1973. Accepted December 13, 1973. Financial support was provided by the National Science Foundation under Grant No. GP-30940X and through the Cornel1 Materials Science Center.
Subpicogram Detection System for Gas Phase Analysis Based upon Atmospheric Pressure Ionization (API) Mass Spectrometry D. I . Carroll, I . Dzidic, R. N. Stillwell, M. G. Horning, and E. C. Horning institute for i / p / d Research. B a y i o r Coilege of Medicine Houston. Texas 66025
A new atmospheric pressure ionization source of simplified and improved design, for use with a modified quadrupole mass spectrometer, is described. Ionization reactions. initiated by electrons from a nickel-63 source, are carried out in a flowing gas stream at atmospheric pressure. The sample is injected directly into the source in common organic solvents. Both positive and negative sample ions result from a complex series of ion molecule reactions. The positive ions are generally M - and M H - ; negative ions are usually ( M - H ) - . These ions are sampled continuously through a 0.025-cm aperture into the mass analyzer high vacuum region and analyzed with a mass analyzer-detector-computer assembly. The source has an active volume of 0.025 c m 3 which is compatible with gas chromatography capillary column requirements. For some biologic samples, such as those from blood or urine, sample preparation consists of solvent extraction. Derivative formation or concentration is often not necessary. I t was found that 150 ferntograms of sample could be easily detected by single ion monitoring.
A preliminary investigation ( I ) of atmospheric pressure ionization (API) mass spectrometry was carried out with a quadrupole mass spectrometer with an ion source of Plasma Chromatography (Franklin GNO Corp.) design ( 2 ) . The instrument in current use, described in this paper, has a source of different design, but has the same form of introduction of ions into the mass analyzer region. Positive or negative ions are generated from sample components through a complex series of ion molecule reactions occurring in the source; primary ionization is due to electrons from a 63Ni foil. In operation, ions and neutral molecules from the reaction chamber enter the mass analyzer through a small aperture (23-micron diameter). In effect, the source is a reaction chamber which is sampled continuously. Horning. M . G Horning. D . I , Carroll I Dzidic. and R N Stillwell. Anal. Chem . 4 5 . 936 (19731 (21 D I . Carroll. R F. Wernlund. and M J. Cohen, U S . Patent ( 1 ) E. C
3.639.757. Feb. 1 . 1972
706
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, M A Y 1974
The primary purpose of the work described here was to find a way of studying ion concentrations within the source in a dynamic fashion, to establish that the response of the analytical system is in the femtogram range, and to examine a n ion profile of organic bases in human urine in order to determine if samples of biologic origin could be studied without a separation step prior to ionization.
EXPERIMENTAL Mass Spectrometer. The mass spectrometer-detector assemand high vacuum system was originally designed (Franklin 0 Corp. j (2) to be used with a Plasma Chromatograph for the purpose of identifying ions present in the drift tube. The basic mass spectrometer is a Finnigan Model 1015, with radio frequency shielding, which has been modified to accept ions from a grounded source and to operate in either the positive or negative ion mode using pulse counting techniques. The vacuum chamber housing is provided with a heating jacket (Briskheat Corp.) for bakeout a t 250 “C. In practice, since ionization occurs external to the vacuum system. bakeout is very rarely required (no cleaning or maintenance within the vacuum system was required during these studies). The vacuum housing is usually maintained a t the same temperature as the source, although it has also been operated for extended periods a t room temperature without degradation of overall system performance. A schematic diagram of the API mass spectrometer is shown in Figure 1. A single aperture ion lens was added to a conventional axial beam electron impact (EI) ionizer to collect the ions conducted from the external source into the high vacuum region through a 25-micron sampling orifice. The E1 ionizer can be used conventionally for calibration of the mass spectrometer. Provision for a quadrupole rod bias voltage, which is required for a grounded ion source, is made by interposing an adjustable voltage supply in both dc ramp lines to the radio frequency generator. The dual voltage supplies impose a selectable dc voltage upon the normal ramp voltages which are then added to the RF voltage ramp. The control voltage for these ramps is supplied by a computer system. The polarity of all lens and rod bias voltages can be reversed to permit negative ion operation without changing the calibration of the mass Spectrometer. The usual 14-stage dynode multiplier has been replaced by a Ekndix Model 4039 Spiratron multiplier. This multiplier is well suited for pulse counting techniques. A special voltage divider is provided for negative ion operation. In this mode, a cathode accelerating potential of +2 kV is provided t o give the negative ions the required initial energy to release secondary electrons. In this case the signal anode is operated a t about +5 kV off ground and
--
7 6 O T O R R v i O
v
TORR-
c
GLASS VAPORIZER GAS EXT
GAS INLET
APERTURE
' ION LENS
1 7
W S S SPECTAOME'ER HIGH VACUUM FLANGE
';-;;-
MULTIPLIER
BLOCK
TT
QUADRUPOLE RODS CALIBRATION ION SOURCE
GLASS CAPi N m 4/ - 1 0
Figure 1. Schematic diagram of API mass spectrometer, showing the inlet system and atmospheric pressure reaction chamber
HEATERSTJ
Cm
25 THERMOCOUPLE
WELL
J
IC,
b-
the pulse signal output is capacitively coupled (0.002 pF, 6 kV) to the amplifier input. A clipping circuit is required to protect the input stages of the amplifier from transients due to arcing. This circuit consists of a 50-ohm resistor in series with two parallel diodes (IN46061 of reversed polarity to ground; in this way, the input transistor stages of the pulse amplifier are protected from occasional arcing. The high voltage supply is a Fluke Model 408B with a n output voltage of k 6 kV. The pulse amplifier system is a Solid State Radiation Corp. Model 1120/1105 Amplifier/Ratemeter. This system has a minimum discriminator setting of 50 p V and a pulse resolution of 20 nanoseconds. The discriminator is normally set at a level of 100-200 pV. The digital output pulse from the ratemeter is 1 volt in amplitude and 10 nanoseconds in width. This pulse is not normally sufficient to operate computer input circuitry, and a pulse stretcher was provided to give a 5-V amplitude, 500-nanosecond positive pulse for computer interfacing. The vacuum system is based on a National Research Corp. Model 3352 pumping system with liquid nitrogen trap and air operated valves. The pumping speed of the system is 900 l./sec; the trap provides additional pumping speed for the solvent vapors and prevents pressure bursts upon injection. The measured presTorr. The actual sure a t the pumping system inlet was 5 X pressure in the ion lens and mass analyzer region was not measured but is believed to be an order of magnitude higher. The measured pressure corresponds to a STP sampling aperture conductance of 0.06 cm3/sec. The mass spectrometer was routinely calibrated through use of the E1 source in a conventional manner. The negative and positive ion mass calibrations were found to be the same within experimental limits of f 0 . 5 amu, and were very stable from day to day. The stability of the instrument was easily monitored by examining solvent and/or background ion spectra. Computer System. A P D P 8 / E computer (Digital Equipment Corp.) with laboratory interface and display scope was used for acquisition and analysis of data from the mass spectrometer. The digital output signal of the SSR counter was connected to a Schmitt trigger input of the interface. The digital-to-analog converter (DAC) output driving the X-axis of the display scope was also used as the input to an operational amplifier circuit to supply a signal of 0 to -10 V to the external scan plug of the mass analyzer circuit. The operation of the system was described previously. The program has been expanded to permit multiple ion detection. The added program alternately switches between each of up to four preselected ions and places the sampled data into one of four 512-word buffers. Source Design. A detailed diagram of the sample inlet system and atmospheric pressure ionization source is shown in Figure 2. The source body is constructed of stainless steel. The gas inlet and outlet ports are Cajon 0.160-cm VCR female fittings welded to the injector. A 0.634-cni male VCR fitting is also welded to the body and serves as the sample inlet fitting. These connections are sealed with nickel gaskets and can be heated to 450 "C. The septum inlet is a modified 0.634-cm VCR female fitting; a 3.8-cm length of 0.16-cm 0.d. stainless steel tubing was silver brazed with an adapter collar into this fitting. A cylindrical GC column septum is placed over the inlet and injections are made through this septum using a Hamilton Corp. Model 7001 (10 fi1) syringe. An aluminum cooling fin is placed on the tubing between the septum and the heated source to decrease septum bleed due to overheating. The injection syringe needle extends 0.6 cm into a standard glass vaporizer (Hamilton Co. parts 18220 and 18234). The internal diameter of the vaporizer tube is 0.25 cm (0.1-cm tubes are also available). The glass inlet parts were silanized.
Figure 2. Detailed drawmg of the inlet system and reaction chamber Samples are injected through the septum with a syringe in a fashion analogous to sample injection in gas chromatography
A 0.75-mCi 63Ki foil, 0.3 cm in width and 1 cm in length was used as the ionization source (New England Nuclear Corp.). This source is approximately one-twentieth of the activity of a standard electron capture source. A 0.0025-cm pinhole aperture (Buckbee Mears Corp.) in a nickel disk (0.95-cm diameter, 0.0025 cm in thickness) serves as the ion sampling orifice. The orifice is sealed to the adapter plate and source body by means of a 0.064cm gold O-ring corner seal. The source and adapter plate are heated by means of three 150-watt-220-volt heating cartridges (Hamilton Co., part 18242). A close-fitting outer housing of insulating material provides thermal stability. A thermocouple inserted into the source flange is used to sense and control the source and adapter plate temperatures. A Thermo-Electric Model 400 proportional temperature controller is used to control the source operating and bakeout temperatures. The source was usually operated a t 200 "C, and raised to 300 "C overnight with normar gas flow for cleaning purposes. Occasionally, persistent samples reTorr a t 350 "C overquired bakeout under vacuum of 5 X night. In this case, a separate pump-down line is used which replaces the VCR septum inlet fitting. Vacuum-tight shut-off valves in the gas inlet and exit lines are used for this purpose. Gas flow rates were measured with a mass flowmeter on the gas exit line (Gow-Mac Instrument Co., Madison, N.J.). A standard GC stainless steel/Teflon flow regulator is used on the inlet gas line; the flow regulator is followed by a cylinder 13X Linde molecular sieve (to remove water and organic materials from the nitrogen carrier gas supply). The design described here can be modified to accept the gas flow from a packed or capillary GC column. Source Characteristics. The source chamber is not electrically isolated, so t h a t the standing current cannot be measured directly. The current due to the 63Ni foil was measured in a separate test, however, using the same foil and analogous cylindrical geometry, and found to be 1.4 X amperes. The current entering the high vacuum region, measured on the first lens element of the mass spectrometer, was found t o be 5.8 x ampere. The total ion current reaching the multiplier was measured by removing the direct current ramp voltage from the quadrupole rods. The observed pulse signal rate was about lo6 cps; the expected rate based on the ion lens current was 3.5 x IO6 cps, indicating about 30% efficiency of the ion lens and rod structure. Reference Samples. Reference samples *ere injected in benzene. It was necessary to employ a set of syringes for different concentration ranges, and blank values were satisfactory only when a syringe was reserved for solvent use alone. The adsorption of samples on glass walls of the syringes was not corrected by ordinary rinsing with fresh solvent; the blank values obtained earlier ( I ) are now known to be due to adsorption on syringe walls rather than on source walls. Biologic Sample. A benzene extract of urine of an individual receiving Ethambutol (who was also a cigarette smoker and coffee drinker) was prepared according to the method of W. G. Stillwell, Hung, Stafford, and M. G. Horning ( 3 ) (described for steroid isolations from plasma or urine) using benzene-ammonium carbonate. Operation. The general operating procedures have been described ( I ) . Since the source chamber described here has a much smaller volume than that used earlier, the flow rates employed (3) W. G. Stillwell, A . Hung, M . Stafford. and M . G Hornlng. A n a / . Lett.. 6, 407 (1973).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, MAY 1974
707
VUL-lPLE QN DETECT C\ 2x10 q BEhZENE d ~ 1 C ~ FdlCCTIILE '~q
I
TIME, SEC
Figure 3. Changes in ion concentration within the source observed after injection of 400 picograms of nicotine in benzene solution. Both MH+ and M + ions are formed from nicotine under these conditions
were 5-10 cm3/min. The volume of injected samples was 1-2 pl; a 1-p1 sample was normally backed by an additional 1 rl of solvent to ensure flushing of the syringe and needle.
RESULTS AND DISCUSSION Reactions in the Source. The complex sequence of reactions which occur in this ion source when an organic compound is injected in benzene, methanol, or chloroform solution was discussed earlier ( 1 ) . It is believed that the initial ionization step involves the nitrogen carrier gas; this is followed by ionization of organic solvent molecules. Another group of reactions results in the formation of ions from the solute by reaction with ions derived from the solvent. Some of the ion molecule reactions which have been recognized are those of proton transfer, charge transfer, and proton abstraction. When benzene is used as a solvent, the ions which have been identified in the source are CsH6+, C12H12+ and small amounts of C6H7+ and C12H13+. If a solute M which acts as a strong base is introduced, proton transfer will occur to yield M H + . If the ionization potential of M is lower than that of benzene, charge transfer may also occur to yield M+ . These effects are shown in Figure 3 for the reactions occurring when a 400-picogram sample of nicotine was introduced in benzene solution. Four ions were monitored; these were C&6+ (76 amu), C6H7' (77 arnu), M' (162 amu), and MH+ (163 amu). The immediate effect was to form Mf and MH+ ions with the disappearance of reactant ions from benzene. As the solute was swept from the chamber over a period of about 40 seconds, the concentration of CsH6+ ions rose, reached a maximum and then decreased in turn as the solvent was swept out by the carrier gas. The concentration of C6H7+ rose slowly (40-50 seconds) to a maximum value which did not change over the next minute as the CsHs+ concentration was decreasing. These concentration changes are interpreted in the following way. The C&7+ ions probably arise through protonation of benzene by H30+ derived from water which is always present in very low concentration in the carrier gas stream. Since nicotine is a stronger base in the gas phase than benzene, the introduction of nicotine results in the disappearance of C6H7+ and the appearance of MHf . Direct charge transfer also occurs leading to M+ , indicating that nicotine has an ionization potential lower than that of benzene. The overall effect is the ionization of nicotine to form both M+ and MH+ . The charge transfer reaction can be suppressed by using conditions which involve only proton transfer. When chloroform containing a trace of ethanol is used as a solvent, the ions formed as reaction intermediates are protonated cluster ions analogous to those formed from water; these ions do not undergo charge transfer reactions, but proton 708
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6 , MAY 1974
transfer will occur if a base which is stronger than ethanol is introduced. Reaction with nicotine under these conditions leads only to M H + . It is possible, therefore, to control the ionization process to some extent by choice of reactant ions. Very few studies have been made of negative ion formation by proton abstraction. Our observations have been limited to studies of the ionization of diphenylhydantoin and the barbiturates by chloride ion; these substances are strong acids in the gas phase, and the usual method of ionization is to inject a chloroform or methylene chloride solution into the system ( I ) . The reaction involves proton transfer from the solute to C1- . Response of Small Samples. Calculations of Ion Lifetimes. An estimate of the total current to be expected a t the ion lens can be made based upon the measured standing current and sampling aperture conductance. A measured standing current of 1.4 X ampere in a 25+1 source volume is equivalent to an ion production rate of 3.5 x 1O1O ions cm-3 sec-'. Under these conditions the ion density in the source is given by the equation:
*
- - - h,n2 + k,P dt where n is the ion density, k l is the recombination rate constant, and kzP the ion production rate of 3.5 X 1O1O ions cm-3 sec-l. Under steady conditions dnldt is zero and the ion density is:
Typical values for kl, for ion-ion and for ion-electron dissociative recombination a t atmospheric pressure are about 10- cm3 sec- ( 4 ) . Substituting this value in Equation 2 gives a steady state ion density of 1.9 X lo8 ions ~ m - ~ . This provides a conductance into the high vacuum region of 1.1 X lo7 ions sec-l, equivalent to 1.8 X ampere. This is in fair agreement with the measured value of 5.8 X 10- l3 ampere and indicates that the assumed recombination rate constant is of the right order. Ion lifetimes in a high pressure source (above about 1 Torr) are often estimated on the basis of ion diffusion losses to the walls. An estimate of the ion lifetime for diffusion losses is given by Equation 3:
(3) where D is the ion diffusion coefficient in cm2 sec-I and L is the relevant chamber dimension for spherical geometry approximation ( 5 ) . For the present source geometry and gas density, the diffusion coefficient should be about 0.10 cm sec- and the ion lifetime based upon diffusion would be about 25 milliseconds (6). From the calculated steady state ion density and assumed reaction rate constant, ion lifetimes based upon recombination would be 1
t --
- k,n
(4)
For an ion density of 1.9 X lo8 ions cm-3 and a recombination rate constant of cm3 sec-l, solution of Equation 4 for this source gives an ion lifetime of 5.4 milliseconds. At these ion densities, and for this form of source design, the diffusion and recombination ion lifetimes are nearly equal and are about 10- second. Lower Limit of Detection. Rate constants for proton and charge transfer reactions range from to l o - $ ( 4 ) E. W. McDaniel, "Colllsion Phenomena in Ionized Gases." S. C . Brown, E d . . J o h n Wiley a n d Sons. New York. N.Y., 1964, p 610 (5) /bid., p 493. ( 6 ) /bid.. p 491.
ETHAMBUTOL 205 MH+
“i“
26-DIMETHYL- I-PYRONE SIM 125 (MH’) FEMTOGRAM SAMPLES
156+ Cl$I2
Figure 5. Positive ion profile observed for a benzene extract of
h
300
human urine for an individual undergohg Ethambutol therapy. The ions at 162 and 163 arnu are due to nicotine a s M + and MH+; ions at 194 and 195 amu are due to caffeine as M f and MH’; the ion at 205 arnu is due to Ethambutol as MH+; the ions at 168 and 174 amu are of unidentified origin I
TIME
Figure 4. Response observed for femtogram samples of 2,6dimethyl-y-pyrone injected in benzene solution. The MH+ ion was monitored: clearance t h r o u g h the source required less than 10 seconds
cm3 molecule-1 sec--l. The sample concentration in the source depends upon the amount of sample injected, the gas flow rate, and upon dynamic factors involving vaporization of solvent and sample in the injector chamber. The equation relating these variables is:
dn, -
+ k,~2,S.
k,nn.
dt
where n, is the sample ion concentration, n the total ion concentration, n, the reactant ion concentration, N, the concentration of sample molecules in the source, and k l and k 3 are the recombination and sample ion production rate constants, respectively. Under steady state conditions d h l d t will be zero and the equation is
k,n,N,
=
k,nn,
(6)
The reactant and sample ion concentrations are related to the total ion concentration by the equation:
n
=
n,
+ n,
(7)
Substituting this identity in Equation 6 and solving for n, yields:
k ,N,n kJ’.
“.=h ,n
+
If k f l S
