Topics in.. ,
Chemical Insfrumenfation
I
Edited by GALEN W. EWING, Setm Hall University, So. Orange, N. J. 07079
These articles are inlended to serve the readers of mrs JOURNAL by calling attention to new developmenla i n the themy, design, or availability of chemical laboratory inslrumentatia, m by presenting useful insighla and ezplanalions of topics lhal are of practical importance to hose who use, m teach the use of, modem instrumenkatia and instrumenla1 techniques. The editm invites correspondence from prospeclive contribulact.
LXIII. Analytical Applications of High Resolution Mass Spectrometry Fred P. Abramson, E. I.duPont deNemours & Co. (Inc.), Instrument Pmducts Division, Monrovia, Calif. 9 10 16 Analytical Applications of High Resolution Mass Spectrometry High resolution mass spectrometry is an especially interesting topic both for its educational content and its analytical value. As an ednrational medium it brings such areas as nuclear binding energies, chemical structures and atomic weights to a common practical use. However, the basis of this article is the latter subjeot: The analytical value of high resolution mass spectrometry. The general principles of mass spew trometry and associated equipment have been discussed in this column before (1, 3); Now the special significance of high resolution mass speetrometry will be given attention. High resolution mass spectrometry is the technique of measuring the exact mass of an ion. A more descriptive name would be exact mass spectrometry. This would contrast it with nominal mass spectrometly, obherwise called low (or medium) resolution. Nominal mass spectrometry measures the whole number mass of an ion; $.g., H 2 0 t = 18, while in exact mass spectrometry HIOt = 18.0106. The purpose of e x s d mass spectrometry is to measure the mass of an ion with enough precision t o unambiguously determine its elemental composition. I n the nominal mass sense, HIO+ = OD+ = H*Cl+% = 88Ar+' = NHI+ = 18, hut with high resolution we find OD+ = 18.0090, H36Cl+2= 17.9883, a'Ar+2 = 17.9838 and NH4+ = 18.0344. Even for simple materials there are a, large number of possible problems which m e resolved by exact mass spectrometry. Until 1958, high resolution mass spew trometry was concerned with determining the exset masses of each nuclide in the periodic table which, when compiled with the isotopic abnndances of each elementoften determined by mass speetrometry
als-yield the atomic weight? generally u e d in chemical calculations. When John Beynon presented a paper in 1958 titled "High Resolution Mass Spectrometry of Organic Materials" (S), he'pioneered a. technique which has been used for the determination of chemical composi-' tions and molecular structures in hundreds of laboratories for thousands of compounds with an ease, accuracy and sensitivity unparalleled in instrumental analysis. Why does higb resolution yield this new information? Table 1 presents the exact masses for a, variety of commonly encountered nuclides. Since each nuclide has a different mass defect (differencefrom nominal mass), high ~.esolutioncan yield the elemental composition without ambiguities far any ion. I n practice, of course, resolution is not infinitely high, but there are sufficiently large mass defects for most commonly encountered isobars (ions of the same nominal mass with different compositions) to make commercially available high resolution mass spectrometers extremely useful. The mass defect for a nuclide arises because, as its protons (mass 1.007825) and neutrons (mass 1.008665) combine into the atomic nucleus, same of their masses are converted into the nuclesr binding energy E by the familiar E = mc2. Since each nuclide has a different combination of protons,neut.rons, and binding energy, its mass is unique. Thus, one can see that high resolution mass speetrometry yields a different type of information than nominal mass spectrometry: the latter tells how many total protons and neutrons there are in any ion and the former tells bow these nucleons are distributed. In other words, a new physical property, mass defect, is observed via high resolution mass specirometry. A similarity to infrared spectra can be drawn. Low resolution ir yields the allowed vibrational levels of a molecule
Dr. Fred P. Abramson received his Ph.11. in physical chemistry from Ohio State University in 1965. He snent two vears as s. nostdoctoral
ventional and tandem mass spectrometers. He joined the Instrument Products Division of E. I. du Pont de Nemoura & Co. Inc. in 1967 (then CEC/Bell& Howell) as a Senior Chemist where he has been head of the mass spectrometer a p plications laboratory for most of that time. His present research interests are the development of new mass spectrometry applications in areas of medicine, biochemistry, toxicology and forensics.
Table 1. Masses of Some Nuclides Encountered in Mass Spectrometric Anolvses~ Nuclide
Mass
= Data, from "Handbook of Chemistry and Physics" (45th ed.), Chemical Rubber Publishing Ca., Cleveland, 1964.
(Continued on page A B 4 )
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Chemical lnstrumentation
Figure 1.
IroboricCompounds.
and high resolution ir provides the rotatiand ceonshnts for those bonds. The terminology of high resolution may be readily understood. The resolution required to separate a doublet a t nominal mass M and an exact mass difference of AM is MIAM. For example, dihydronaphthalene and phthalazine require a resolution of 5167 (see Fig. 1). These ions have no natural line width (the mass of an ion is only subject to the inaccuracies of the uncertainty principle) 80 the breadth of a peak is representative of the analyzer performance. A resolution specification is generally indicated by the valley between two peaks of equal size with a given MIAM for the pair. Figure 1 shows the 10% valley definition using the dihydronaphthalene/phthalazine doublet to demonstrate a. resolution of 5167. The most important specification for molecular formula. computation is the mass measurement accuracy. The mass measurement accuracyis affectedby the resolution to the extent that unresolved mass multiplets will give highly erroneous results due to the substantid peak distortions which result. For this reason, exact mass measurements are generdly made at a resolution of 10,000 or greater. At this resolution, mass measurements with a standard deviation of 1 part in 500,000 are achievable. Thus, much greater accuracy for mass measurement is available than the mass resolution would indicate. This 50-fold improvement in accuracy can only be obtained by careful analysis of the unknown peak profile with respect to some reference mass peak profile. Indeed, if the peak positions are measured to 'Iwthof the width, there can he no distortion of one of the two peaks caused by an unresolved multiplet or instrumental effects. Extreme care in design is necessary to provide this high mass measurement accuracy. High resolution mass spectrometers differ from their nominal mass counterparts in most of their components. The ion source is designed to produce an especially intense beam of properly focused ions to admit to the analyzer through an adjustable object slit. This exbra intensity is beneficial because the beam currents during high resolution operation may he orders of magnitude smaller than in nominal mass operstibn. As aresult, most high resolution mass spectrometers d l h v e excellent sensitivity when operated well below their resolution limits. The traditional approach to high resolution analyzers is to make them very large. (Continued on page A2881
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Chemical instrumentation Nearly all high resolution instruments sold until recently had magnetic sector radii of at least 12 in., which was one of the factors leading to their $100,000+ price tag (Fig. 2). Limitations of theory and design prevented smaller instruments from attaining the required performance. Modern mass spectrometry theory and design have now produced analyzers for high resolution mass spectrometers of less than half the size and price of older units (Fig. 2). Thus, a new era of convenience, utility and affordability of exact mass spectrometry is here. A question often arises whether single or double focusing mass spectrometers are to he preferred for exact mass measurement. Beynon (4) points out: "In a single-focusing machine however, the most accurate measurements must be restricted to molecular ions, which are formed in the ionization chamber without an appreciable kinetic energy. Since a. (single focusing) sector-type mass spectrometer is not velocity focusing, ions which enter the machine with kinetic energy will he deflected in the magnetic field on a larger radius of curvature than ions of the same mass which have no kinetic energy of formation, and will therefore he recorded as having an increased mass." Two equations are required to point out the value of douhle-focusing analyzers. The resolution of any mass spectrometer is Resolving Power
=
DM 2W
(1)
where D is the dispersion of the analyzer design-that is spatially how far apart are two masses whose mean mass is M-nd W is the beam width. The important term here is W which obeys the following equation:
W
=
mS
+ kla + krP + knu' + kanP
+ knP2 + . . .
(2)
where m is the magnification of the an* lyzer (as you can see, the smaller the magnification, the Larger a source slit S, can be used for a given resolution, hence, greater sensitivity), ar is the angular dispersion and p is the velocity dispersion of the ions and the k's are the aberration coefficients; that is they indicate how much effect a given angular or energy spread will have. I n a single-focusing analyzer, k, = 0 and all the other terms have finite values. Thus, energy spread has a linear effect on beam width. In a douhle-focusing mass spectrometer k2 = 0 and in a seeand-order corrected double-focusing mass spectrom= 0 as well, so that the eter k,, = k , ~= angular and energy spreads in the beam have no effects on peak shape or position. A second-order corrected douhle-focusing analyzer therefore can accept 8. wide range of angles and energies and not show a corresponding beam width increase. I t is for these reasons that ttn overwhelming majority of exact mass spectrometers ever
( C a t h u e d on page A288)
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Chemical Instrumentation produced have been, and still are, of double-focusing designs. I t is not just sufficient to construct an appropriate analyaer. The electronics and vacuum characteristics of an exact msss spectrometer must also he given special care. Because o f , the higher resolution required, the vacuum must he maintained a t an especially low value so that there will be no ion scatter to broaden the resolved ion beam. Because of t,he part-per-million character of exact mass measurements, the magnetic field stability as well as the mechanical integrity of the analyzer must be free of variations dawn to that low level. I n summary, an exact mass spectrometer analyzer is one which has a number of special characteristics all of which are necessary for proper performance. There are three methods for the delcrmination of exact mass. In all cases, some reference compound with exactly known mass is introduced with the unknown sample. The location of each msss must he measured relative to some other mess because the parameters of the mass spectrometer, voltage, and magnetic field cannot be measured with sufficient accuracy to mzke n direct measurement. I n the first of there methods, the mass spectrometer is scanned and the positions or emergence times of the known masses are used to determine the masses of the unknown. For small mass regions, 1-10 masses, this esn be done with any analog recording device, suoh as an oscillngraph. Entire msss spectra are rarely taken this way since such a record would be several hundred feet long. When entire scans are taken they are read into a data acquisition system and subsequently processed. The second method is by analyzing a photographic plate without scanning. The photoplate integrates the charge a t each mass and, after development,, the positions of the lines (about 10 Nm width on a 2.5 cm photoplate) are read and converted to mass. The third t,echnique is called peak matching. Here the mass of an unknown peak is compared with a reference mass directly by the me of a precision potentiometer which varies the accelerating voltage of the mass spectrometer and thus the mass in regisber (Fig. 4). By carefully superimposing the peak positions of t,he known and unknown masses, very high accuracy for an exact measurement is obtainable, generally higher than far the first two techniques. The goal of exact mass spectrometry is not merely to obtain an exact mass number but to determine the elemental composition of the ion of interest. There are several publications which present tables of methods for performing such cdculations (6-8) mamlally and a variety of on-line methods too numerous to list. The parameters are the exact mass, the d o w e d tolerance and how many atoms of which elements are anticipated. Any number of elements in any quantity may be examined hut the amount of computing time and the ambiguity of answers will both go down if reasonable parameters are entered. (Continued on page A290)
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Chemical Instrumentation The output from exset mass spectrometers is int,ended to be very high in qualitative content hut due to the reduced intensities, high resolution spectra usually have less quantitative information than desired. For this, and other reasons a s well, high resolution is not often the first technique used on s. sample. The nominal maas spectrum of a compound will be taken and analyzed init,ially. When a
Figure 2.
problem in ident.ification exists, one or more peaks in that spectrum may be subjected to exact mass measurement to give a more definitive result. Because of this typical sequence, low followed by high resolution analysis, it is import,ant that the mass spectrometer be capable of rapid conversion between high and low resolving power, far this may be done many times in a given day. The applications of exact mass spectrometry in orgenic chemistry (or inorganic chemistry if the materials are vola-
Du Pont 21-1 1 0 High Resolution Moss Spectrometer. Bored on a 1 2 in. radius magnetic onolyrer, it fir4 sold in 1 9 5 9 and could achieve 30,000 rerolring power. Its price was $1 25,000.
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tilieable) fall into three basic areas: (I) identification of unknowns, (11) verifications of nynthesis, (111) determinat,ition of structures via group losses. Looking at a. 6 month span of the Journal of the American Chemicd Societ,y yields examples of each type of me. (I) Often a novel material will he encountered. Its novelty may he very important if it is a. new sort of compound, or iaola.ted from a novel source. Sims, et al. (.9) have isolated a structural variant from ~,&renica .vaeifiea,~a red dm. .- . which is the first natural compound which contains both bromine and chlorine. High resolution measurements gave the formula C,.,&t01BrnCl confirming their finding. Much of chemistry is involved in new synthetic methods. In biological and biomedical applications, often metabolites are isolated which have never been observed before (10). Exact mass spectrometry is a very valuable tool in such studies, as the numerous high resolution instruments in pharmaceutical companies will attest. (11) Often new classes of compounds are being created (where technique I is valuable) or else new routes to more familiar species. Corey, el al. (11) have prepared 21 compoundssame synthetic intermediates and some final products-and have examined the molecular ions of each to verify that their synthetic methods are producing the anticipatedproduct. Exact mass spectrometry supplements chromat,ographic analysis, elemental analysis, (Continued on page A29S)
Chemical Insfrumentation
Figure 3. Du Pont 21-492 High Resolution Mas. Spectrometer. Bored on o new 4 in. radius mognetic andyrer, this 15,000 resolving power instrument was introduced in 1970 and relkfor $52,500.
ir and nmr data which is the best procedure in modern instrumental analysis. Each analysis provides unique information (ex., cis-trans olefins by ir, double bond position from nmr, etc.) and the greater the number of inputs to the analysis, the less tho p o sihilit,y of error. (111) Many of the detailed physical organic mass spectrometry studies which have been carried out over the years have been performed with the philosophy that, if the mass spectrum of n group of related compounds could be very well described, t,hm s. new member of that class could be unambiguously identified without m y standard being required. A number of methods are used for this purpose; appearance potential measurements, metastable reactions and correlation of group losses with structure. Benyon comments on the importance of the last method (3, p. 345). ~
~
~
~
"It will certainly never be possible to predict breakdown patterns leading to the mass spectra, of organic compounds
Figure 4. Mars Mearvrement Apparatus. A reference mass A and an unknown m a s B are brought into coincidence b y adjusting the IMB MA)/MA contml while the circuit ir alternately sweeping over the A peak marr and the B peak maxr. For maximum accuracy, the two peak heighh are adjusted to be equal by attenuating the lorger peak with the amplitude control. The unknown marr MB = M n [ i IMs - Mnl/M~l.
+
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until the compositions of fragment ions can he obtained with certainty.. many of the ions in the high resolution spectra are of unexpected composition and it is hoped that the greater detail will, by making clear the composition of the ion, and often the path by which it hsl, been formed, make correlations of the structure of molecules and their mass spedrs. more complete!'
.
In a. recent article of mass spectrometry of nucleic acid components, Lawson, et al. (18) use the exset mass measurement of fragment ions frequently to examine the relationships of structure and spectra for derivatives of such compounds as 2'-deoxyuridine 5'-phosphate and adenosine 3',.5'-cyclic monophosphate. To predict the structures of fragment ions from a molecule of elemental composition ClnHsr N5OaPSis would be a very difficult endeavor without the assistance of exact mass spectrometry. The applicstions of high resolution mass spectrometry are very broad, indeed. An exact mass determination is probably the single most informative instrumental measurement of an unknown compound. As such it represents a very valuable asset to the chemist and, because of the continuing importance of an malytical determination as fundamental as a. molecular formula, high resolution mas3 spectrometry will continue to play s significant role in chemical instrumentation. The reader is referred to the literature cited (9-18) for more detail about the utdization of the exact mass data. Also,
a recent chapter by Biemmn (13) details many other aspects of the interpretation and data presentation of high resolution mass spectra which cannot be presented here. Further, the biennial review chapter on mass spectrometry in Analytical Chemistry Analytical Reviews (14) serves as a. good source for high resolution studies.
(21 Ewma. G. W., J. CHSM. EDVC., 46, A69, A149, A233 (1969). (3) B ~ m o nJ. . ti.. in "Advances in Maas Spectrometry" (Editor: W ~ ~ n n o sJ.. D.). Pergamon,Oxford,1959,Val.l,p.328. (41 B e w o n , J. H.."Mass Spectrometry and ita Applications t o Organic Chemistry." Elsevier, Amsterdam. 1960, p.41.
. Anal. Chcm.. 35, 2146 (1963). (61 K m n n l c ~E.,
Manufacturers
(7) LEoEneEno, J.. "Computation of Moleaular Formulas for Mass Spectrometry," Aolden-Day. SanFranoisoo. 1964.
AEI Scientific Apparatus, Inc. 500 Executive Blvd. Elmsford, N. Y. 10523 E. I. du Pant de Nemours $ Co., Inc 1500 S. Shamrock Ave. Monrovia, Calif. 91016 Hitschi, via. Perkin-Elmer Corp. 800 S. Main St. Norwalk, Conn. 06852 JEOL U.S.A., Inc. 235 Birchwood Ave. Cranford, N. J. 07016 LKB Instruments, Inc. 12221 Parklawn Drive Rockville, Md. 20852 Varim Associates 611 Hansen Way Pitlo Alto, Calif. 94303
(81 TnlrrrloLrFF. D. D., W n o s w o n ~ x ,P. A,, A N D SomlasLER, D. 0.. "C. ti. 0,NMass s n d Abundance Table," Shell Development Co.. Emeryville, Calif.. 1965.
. D.. A m Tnncr;n. W. F.,Sciencc. (10) B n n c ~ G. 173,544 (1971). (11) CORET,E. J., ERICH~ON, B. W.. A N D NOYORI, R.. J . Amer. Chem. Soc.. 93, 1725 (19711. LAWSON. A.M.. STILWELL. R. N.. TICXER. M. M.. TBOBOTAMA, K.. AND MCCLOBKEI, J. A,. J . Amer. Chem. Soc.. 93, 1015 (19711. (131 BIBMANN.K., in " T o ~ i o sin Organic Mass Spectrometry" (Editor: BURLINGAME. A. L.1, Vol. 8, "Advsnoesin Anslytical Chemiatry and Inatrumentation." Wileu. N e v Ymk; 1970, p. 185.
Literature Cited (1) W ~ e n s ~ mS.. E., AND AIKENS. D. A,. J. C n e ~ . E n u c . ,41, A75, A153 (19641.
(14) A n d . Chcm., (1968) eto.
42, 169R (19701: 40, 273R
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