2288
Anal. Chem. 1984, 56,2288-2290
measurement providing several “lock masses”can be found in the spectrum of the unknown. Registry No. LY135809,91759-08-9; LY146957,91797-56-7; H(OCH2CH2),0H, 25322-68-3; ceftazidime,72558-82-8;glycerol, 56-81-5.
LITERATURE CITED (1) Gllilam, J. M.; Landls, P. W.; Occoiowitz, J. L. Anal. Chem. 1983, 55, 1531-1532. (2) Surnam, D. J.; Vickerman, J. C. J . Chem. SOC.,Chem. Commun. 1981, 7 , 324-325.
(3) Barber, M.; Bordoli, R. S.; Sedgewick, R. D.; Tyler, A. N. J. Chem. Soc., Chem. Commun. 1981, 7 , 325-327. (4) Rinehart, K. L., Jr. Science (Washlngton, D.C.) 1982, 278, 254-260. ( 5 ) Van Langenkove, A.; Costello, C. E.; Chen, H.-F.; Biller, J. E.; Blemann, K. Annu. Conf. Mass Spectrom. Allied Top. 1082, 558-559. (6) Morgan, R. P.: Reed, M. L. Org. Mass Spectrom. 1082, 77, 537. (7) Kimble, E. J. I n “High Performance Mass Spectrometry: Chemical Appllcations”; Gross, M. L., Ed.; Amerlcan Chemical Society: Washington, DC, 1978. (8) Goad, L. J.; Prescott, C. M.; Rose, M. E. Org. Mass Spectrom. 1084, 79, 101-104.
RECEIVED for review March 26,1984. Accepted June 25,1984.
Surface Precipitation Technique in Fast Atom Bombardment Mass Spectrometry Mei-Yi Zhang and Xi-Yun Liang* Institute of Chemistry, Academia Sinica, Beijing, The People’s Republic of China
Yu-Yi Chen and Xiao-Guang Liang Institute of Photographic Chemistry, Academia Sinica, Beijing, The People’s Republic of China
A novel surface preclpltatlontechnique was used to obtaln the fast atom bombardment (FAB) mass spectra of substltuted tetraphenylporphyrln (TPP) and thelr metal complexes. The method consists In dissolving the sample In chloroform and alowlng the solution to evaporate on nonoxynol used as the vlscous matrlx. It Is useful In obtaining FAB mass spectra of solvent-soluble lnvolatlle compounds which are not soluble In the matrk material. Correlatlon of the lon lntensltles wlth the “surface concentratlon” of the sample Indicated that surface mobility of the sample partlcles Is probably responsible for malntalnlng a steady Ion current for an extended perlod of time.
Since ita first introduction (1,2), fast atom bombardment mass spectrometry (FABMS) has developed into a powerful tool for obtaining mass spectral information of large polar molecules (3). Although the mechanism for the ionization process is still not clear (3) and controversy over the necessity of neutrality of the bombarding particles in the process still persisted ( 4 ) ,the importance of the viscous liquid matrix is almost universally recognized. It is generally believed that solubility of the organic sample in the liquid matrix, usually glycerol, is essential to obtain useful FAB mass spectra (5,s). As a result, the FAB technique is used most widely for biomedical and biochemical samples which are hydrophilic and, therefore, soluble in glycerol. While the usefulness of FABMS in these fields should not be understated, there are a great many high molecular weight compounds which are not soluble in glycerol, thioglycerol (7),and the like (8), and therefore, precluded from taking advantage of the method. I t is to this problem that our report is addressed. In our attempta to obtain the mass spectra of tetraphenylporphyrin (TPP) and its derivatives, we found that dissolution of the sample in the liquid matrix is not necessary to obtain a good FAB mass spectrum; by precipitating the sample in situ on the surface of a viscous matrix, we have been able to obtain good quality FAB mass spectra of all the TPP derivatives at 0003-2700/84/0356-2288$01.50/0
our disposal. The method we report here is straightforward and promises to be generally applicable.
EXPERIMENTAL SECTION The nonpolar surfactant nonoxynol (a poly(ethy1ene glycol) p-nonylphenylether) (I), obtainable from GAF Co. of Japan, was used as the liquid matrix. It was applied pure on the surface of the FAB target probe. The TPP sample was dissolved in chloroform and 2 pL of the solution was transferred to the surface of the liquid matrix with a microsyringe. The loaded probe was then inserted into the forevacuum lock, where the chloroform was evaporated. After 1 to 2 min, the probe was inserted into the ionization chamber for bombardment in the usual manner. The mass spectra were obtained on a AEI MS-50 mass spectrometer equipped with a standard FAB source from the same company and a fast atom gun from Ion Tech. Inc. Argon atoms of about 7-keV energy were used as the bombarding atoms. Spectra of compounds of molecular weights less than 900 were run at acceleration voltages of 8 kV and those above 900 at 6 kV. Only positive ion mass spectra were measured in all cases. The molecular ion intensity at various sample “surface concentrations”were determined as follows: A saturated solution of TPP was prepared and its concentration determined by differential weighing. Solutions of different concentrations were prepared from the saturated solution; a 2-pL aliquot of each solution was introduced into the mass spectrometer in turn as described above. In cases where higher sample concentration than saturation is required, multiple injections on the probe surface were used. The single ion monitoring technique was used to follow the change of molecular ion intensity with sample “surface concentration”. The absolute intensities after subtracting the background signal were used for correlation. The term ”surface concentration”is used with qualification, since it merely denotes the amount of sample per unit area of the probe surface, rather than the concentration of a real solution.
RESULTS AND DISCUSSION Using the simple and straightforward method described above, the FAB mass spectra of TPP (111)and ita derivatives with various substituents on the peripheral phenyl rings and its complexes with metals were obtained. To illustrate the molecular weight range covered in our experiments the high mass end of the FAB spectra of three alkoxy-substituted 0 1984 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
2289
889 IY+HI+
(VI
I
Lit 676 I Y + H I +
,I
1043 IM+H)+
(VIIJ
(d 1
IC1 Flgure 1. FAB mass spectra of
TPP derivatives and metal complexes.
I B'5'Y+H'* Substllrtnt
(VI11
R-H
Metal
Y-Cr
1~1. Wt. 815
TPP's is shown in Figure 1. These spectra showed clearly the protonated molecular ions at m/z 721 (IV), 889 (V), and 1043 (VI), respectively. No aggregate ion with the alkali metals is observed in all cases. Due to the insolubility in glycerol it was impossible to obtain any useful FAB mass spectrum of the TPP derivatives by dissolution or mixing in the usual manner. Barber and co-worker reported the use of Trition XlOO (11)as an additive to increase the solubility of chlorophyll a in glycerol and succeeded in producing its FABMS (5). However, such modification and other dispersion methods, including prolonged grinding, ultrasonic agitation in various liquid substrates, and even precipitation using the same solvent-matrix combination as in our experiments, give only extremely weak signal even in the most favorable case. In Figure 2, trace B shows a section of the best FAB mass spectrum of TPP itself obtained by prolonged grinding of the sample in a mortar with nonoxynol substrate. The quasimolecular ion at m / z 615 is barely distinguishable among the background signals of the substrate. The spectrum obtained by surface precipitation is shown in trace A; the same section of the spectrum of the pure substrate is also shown in trace C of the same figure for comparison. It is worthwhile to note that in traces B and C the peak at m / z 617 belongs to the series of protonated molecular ions of the nonoxynol substrate, rather than to the sample. In addition, trying to disperse the TPP sample in glycerol by any means failed to yield any sample signal. The success of the method, we believe, depends on the way microparticles are formed on the substrate surface. We postulate that on injection of the sample solution onto the substrate surface, two separate layers are formed in the first instant. As the two miscible liquids diffused into each other, the sample precipitates out in the form of minute microparticles, the size of which depends on the speed at which they are formed. After the volatile solvent has been removed rapidly under vacuum, the sample is eventually left on the substrate surface as a finely dispersed emulsion layer. The sample in this highly divided state has a very large surface area and is, therefore, susceptible to desorptive ionization by fast atom bombardment. The sequence of the process is
A comparison of FAB mass spectra of TPP using surface preclpltatlon and direct mixing.
Flgure 2.
VACUUY
__.___ . . .. ......... . . ... .... . .. ..... . . . .
A&&&&
-
.._... ~
-*
I
b
-
~
.
___f
E
The process of surface precipitation.
Flgure 3.
I 100 200 SAYPLf A M O U N T
300
IN!.)
Flgure 4. Correlation of protonated molecular ion intensity with the sample surface "concentration".
summarized in Figure 3. It is not clear though how and when the proton is transferred to the molecule being ionized: Whether the proton is abstracted from another sample
2290
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 614
I 893 l M + H l *
u.,k d L
900
Flgure 5. Single ion monitoring of protonated molecular ion before (5a) and after (5b) the intensity maximum in Flgure 4.
molecule or from one of the surrounding substrate molecules. Diffusion of the sample molecule from the bulk of the liquid matrix to replenish the solute-depleted surface has been proposed ( 4 ) to account for the steady and long lasting ion current obtainablefrom the FAB of soluble samples. Recently, Ross et al. reported the use of liquid metal as the substrate for obtaining SIMS of organic compounds and suggested that surface mobility of the sample particles can also replenish the deplete area and maintain a steady ion beam for a long time. We believe that a similar surface migration mechanism is operative in the surface emulsion from precipitation, as we can also maintain a steady secondary ion current for more than 30 min. The hypothesis seems also to be in agreement with the result of correlating sample “surface concentration” with the intensity of the protonated molecular ion peak ( n / z 615) shown in Figure 4. The intensity of the protonated molecular ion peak increases with the amount of sample applied up to a maximum (at a sample dosage of ca. 75 pg) and then falls off again. This is indicative of the existence of an optimal “surface concentration” of the sample particles, above which, the surface mobility of them will be so restricted that replenishment of the depleted area is retarded. As a result, any further increase in sample quantity actually reduces the ion intensity rather than enhances it. Furthermore repeated scanning over the pronated molecular ion showed a very steady ion intensity over a long period of time up to the point where sample surface “concentration”is optimal (Figure 5a). While in the cage of over dosage, the intensity tends to decrease steadily with time (Figure 5b) confirming again that replenishment by surface convection is slower than sample ionization and desorption by FAB. Although the surface precipitation technique described above is most useful for samples which are completely insoluble in the liquid matrix, e.g., VII. Depositing the sample on the matrix surface as a solution in volatile solvent is useful for both soluble and insoluble materials. As an example, the mass spectrum of chlorophyll a obtained by this technique is shown in Figure 6. Although the same have been obtained by the dissolution method using Triton XloO as a solubilizing agent (5), however, it does demonstrate the general applicability of the method we employed. As a result, the operation of sample introduction in FABMS can be greatly simplified
830
680
605
Flgure 6. The FAB mass spectrum of chlorophyll a obtained by surface deposition of the sample on the target probe.
and applied routinely in day to day work. In conclusion, by choosing nonoxynol as the liquid matrix and using a very simple on-probe precipitation technique, we have obtained the FAB mass spectra of tetraphenylporphyrin derivatives and metal complexes. We have shown by our example that dissolution of the organic sample in the matrix is not essential in obtaining good FAB mass spectra. Provided that we can find a volatile solvent which can dissolve the sample and at the same time be miscible with the liquid matrix, we stand a good chance of running a successful FAB experiment. Such a requirement is much less stringent than having to look for a viscous liquid substrate to dissolve the sample, as liquids that can qualify as substrate material are so few in number. Furthermore, the method does not require any special instrumentation nor modification of standard equipment. It is hoped that by making our findings public, more solvent-matrix pairs can be unveiled and more classes of compounds studied, making FAB a truly powerful and versatile analytical tool for large molecules.
ACKNOWLEDGMENT The authors thank Lui Shueh-lan for supplying us with the poly(ethy1ene glycol) nonylphenyl ether and Ho Hui-chu for her gift of chlorophyll a. Registry No. 111,917-23-7;IV, 87719-17-3; V, 91410-72-9; VI, 91410-73-0; VII, 14172-91-9;chlorophyll a, 479-61-8; nonoxynol, 26027-38-3.
LITERATURE CITED (1) Barber, M.; Bordoli. R. S.;Sedwick, R. D.;Tyler, A. N. J. Chem. SOC., Chem. Commun. 1981; 325-327. (2) . . Surmann, D.; Vickermann. J. C. J. Chem. SOC., Chem. Commun. 1981, 324-325. (3) Williams, D. H.; Bradley, C.; Bojesen, G.; Santikarn, S.;Taylor, L. C. E. J. Am. Chem. SOC. 1981, 103, 5700-5704. (4) Aberth, W.; Straub, K. M.; Burllngame, A. L. Anal. Chem. 1982, 5 4 , 2029-2034. --- (5) Barber, M.; Bordoil, R. S.;Elliott, G. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 54, 645A-657A. (6) Ross, M. M.; Colton, R. J. Anal. Chem. 1983, 55, 1170-1171. (7) Rottschaefer, S.;Roberts, G. The 31st Annual Conference on Mass Spectrometry and Allied Toplcs, p 286. (6) Hlguchi, T.; Sparkman, D.; Aoyama, T.; Arita. M.; Iwamori, M.; Nagai, Y. The 31st Annual Conference on Mass Spectrometry and Allied TopIcs, p 292.
__
RECEIVED for review February 21, 1984. Accepted May 29, 1984.
