Anal. Chem. 1966, 58, 1661-1664
1661
Dependence of Ion Formation upon the Ionic Additive in Thermospray Liquid Chromatography/Negative Ion Mass Spectrometry Carol E. Parker,* Richard W. Smith, Simon J. Gaskell,' and Maurice M. Bursey2 Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
The nature of negative ion formation from organic compounds-by anion attachment or by proton ab8tractlon-h thermospray LWMS may be predkted on the basis of gakphase acidities. From a general table of gasphase acidities known for representatives of most common Ion functional group classes, the thermochemlstry of [M formation can be esthnated for mixtures of many polar compounds and ionic addntves. Predictions are fulfilled for several model analytes: analogues of dlethylstllbestrol, pesticide metabolites, cortlcosterokls, and chkwamphenkol propionate, with different Ionic additives.
- lr
It has recently been suggested (1,2) and then demonstrated (3, 4) that gas-phase processes play a role in thermospray liquid chromatography/mass spectrometry (TSP LC/MS). The role may not be a universal one, for substances that exist as ions in the eluant may form unsolvated ions in TSP by stripping solvent from the solvated ion (5);but for species that elute as neutrals, the ultimate process can be gas-phase ionization. In such cases positive-ion spectra of analytes in 0.1 M ammonium acetate, for example, have been widely noted to compare closely with ammonia chemical ionization (CI) spectra. Negative-ion TSP spectra have been less widely used than positive-ion TSP spectra. In negative-ion TSP of neutrals without an ionizing filament, there are two important mechanisms for negative ion formation: proton transfer and anion attachment; in "filament-on" TSP, a third mechanism, electron capture, is possible. This multiplicity can create some difficulty in assignment of molecular weights to unknowns. Nevertheless a few examples have now been found in which formation of [M - 11- ions from analytes follows gas-phase reactivity in TSP (3) and direct liquid introduction (DLI) (6), and so a basis exists from which to extend arguments to predict when proton transfer is important and when anion attachment is important in practical negative-ion LC/MS.
PRINCIPLE Several examples of pairs of acids or bases have been examined by positive and negative TSP (3) in which the equilibrium lies well to one side in solution but to the other in the gas phase; in each case gas-phase reactivity was observed. This study provides more detailed consideration of the ionization mechanism in negative-ion TSP processes. Therefore, our discussion relates to the gas phase. We assume that the molecule studied by LC/MS contains hydrogen and call it H A we call the anion of the additive B-. 'On leave from the Tenovus Institute for Cancer Research, University of Wales Colle e of Medicine, Cardiff, UK. 2 0 n leave from the dlliam Rand Kenan, Jr., Laboratory of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27514. This
article not subject to U.S. Copyright.
When HA and B-collide, there are two competitive processes that can lead to products, eq 1 and 2.
+ BHA + BHA
-+
-+
AHB-
A-
HB
(1) (2)
For almost all polar molecules studied by TSP LC/MS, eq 1is the formation of a hydrogen bond. Thermochemically this reaction is always favorable: AH," is the value of the strength of the hydrogen bond between HA and B-. The strength of gas-phase hydrogen bonds has been found to be 41-77 kJ mol-' for bonding to chloride (7), 97-139 kJ mol-' for bonding to fluoride (B), 123-128 kJ mol-' for bonding to acetate (9),and 80-92 kJ mol-' for bonding to alkoxide (10). These values are taken from the few available examples, in the case of acetate, only two; the ranges will be broader when more examples have been studied. Other factors being equal, the greater the exothermicity of the clustering reaction, the deeper the potential well, and the more likely that collisional (or radiative) stabilization will permit its observation. On the other hand, a cluster with energy inadequate to dissociate back into reactants may still have enough energy for a rearrangement. To review the competitive deprotonation process, eq 2, a detailed examination of available data relevant to proton transfer is required. It is possible to generalize about the gas-phase acidity of molecules with typical organic functional groups, thanks to a recently available collection of such data (11). The gas-phase acidity of a molecule HA is the Gibbs free energy of the hypothetical gas-phase reaction, eq 3. This confusing nomenclature-as acids become stronger the numerical values of their gas-phase acidities decrease-is unfortunately standard in the literature of gas-phase negative not AGIO (12, ions, though earlier the term represented MIo, 13). Those unaccustomed to it will find that a parallel argument can be constructed from the gas-phase basicities of the conjugate bases of these acids. The AH/ values in Tables I and I1 are then the proton affinities of the anions. In this case the definition makes numerical sense: as conjugate bases become stronger the numerical values of their gas-phase basicities also increase.
HA
-
H+ + A-
(3)
The gas phase acidity of HA may be compared with the gas-phase acidity of another acid, HB whose conjugate base, B-, competes with A- for the proton in eq 2. Ideally the comparison should be the determination of the position of equilibrium, i.e., the calculation of AG,", for eq 2. This AG," is equal to gas-phase acidity of HA minus the gas-phase acidity of HB. Because of the unfortunate standard definition of gas-phase acidity, the free energy change has a negative value, and is favorable, when the gas-phase acidity of HB is greater than that of HA. To recapitulate, the smaller the numerical value of the gas-phase acidity, the stronger HA is as an acid and the weaker A- is as a base.
Published 1986 by the
American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8 , JULY 1988
1662
SZY
100\
80
.-*
3F
100
VI1
VI
-
c,
fn
s -C
60-
I,
Q)
.-5
-m
c, P)
K
2o 0
140
180
3e5
100
180
IX
220
260
140
3 0 0 3 4 0
m/z Flgure 1. Negative-ion TSP mass spectra of analogues of stilbestrol with 0.1
Table I. Gas-Phase Acidities of Typical Organic Functional Groups (I1 ) HA
AH,"(eq 5), kJ mol-'a
CH, PhCHB p-NOZPhCH3 PhzCHz
1743 1571 1477 1525
3"
1689 1686-1661b
RNH, (CH3fDH PhNHz P-NOZPhNH, benzene
HCCH CHSOH RCHgOH RzCHOH, R3COH PhCHzOH CH3SOCH3
1657 1536 1447 1669 1571 1587 1573-1556' 1564-1544 1545 1559
2
100
Vlll
\
3 0 0 3 4 0
m/z
m/z
140
260
220
HA
AH,"( e s 5), kJ mol-1a
RCOCH, enol 1544-1536 RCHO enol 1531-1515 PhCOCH, 1520 RzC=NOH
1536-1527
PhSOZCHzR CHaSH RSH PhSH
1527-1515 1502 1490-1473* 1431
RCHZNOz 1501 PhCONHCH3 1476 (RCO)zNH 1452-1443 PhOH p-NOzPhOH CH3COOH CFSCOOH PhCOOH
1464 1379 1458 1350 1418
CHSCN 1557 RCHXN 1565 PhCHzCN 1478 p-NOZPhCH,CN 1393 Mostly 1 1 0 kJ mol-'. *Decreasing AH,''for larger R.
If the ionization process in TSP is gas phase, then the additive B-will form A-from H A by proton abstraction when H B has a numerically higher gas-phase acidity.
180
220
260
300
340
m/z M ammonium acetate as ionizing additive.
Table 11. Gaseous Acidities of Selected Mineral Acids (I1 j
AH,''(eq l), compound
kJ mol-'
compound
AHF' (eq 11, kJ mol-'
HOH HOOH HF HSH
1635 1566 1554 1479 1416
HC1 HNO, HBr HI
1395 1358 1354 1315
HNO,
However, AG," values for eq 3 are not available for more than a few compounds. We will assume that AS," values for eq 3 for different HA are similar to each other, and 50 the side to which equilibrium lies in the TSP plasma can be calculated from AH,"values. A summary of A",O values for organic compounds is given in Table I, and a summary for possible mineral acids whose salts might be used as ionic additives is given in ';'able 11. The principle then is as follows: (i) When eq 2 is endothermic, it is inoperative; H A forms AHB-only. (ii) When eq 2 is exothermic, H A forms A-;A-will be much more abundant than AHB- if AHB- fragments by rearrangement or if the pressure is inadequate to stabilize it collisionally. (It is conceivable, though unlikely (14), that an additive anion, B-,of intermediate basicity could react with a molecule with two greatly separated acid sites to form AHB-at the site of lower acidity, rather than A- a t the more acid site.) These arguments are similar to, but more explicit than, those recently advanced for positive-ion TSP LC/MS reactivity (15).
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
EXPERIMENTAL SECTION The metribuzin metabolites, available from a previous study (16),were chosen to illustrate the origin of [M - 11- peaks, since their TSP spectra contain only [M - 11- peaks when they elute in 0.1 M ammonium acetate. Hydrocortisone and corticosterone (Sigma Chemical Co., Inc., St. Louis, MO), on the other hand, give only [M + Ac]- peaks under the same conditions. Hexestrol, E-diethylstilbestrol, E$-dienestrol, and indenestrol A were selected because their spectra contain varying ratios of [M - 11-and [M Ac]- peaks when they elute in 0.1 M ammonium acetate. The hexestrol and E-diethylstilbestrol were purchased from Sigma Chemical Co., St. Louis, MO; the others were gifts of Manfred Metzler, University of Wiirzburg, FRG. Chloramphenicol propionate was selected because with 0.1 M ammonium acetate its spectrum contains [M - 11-, [M Cll-, and [M Ac]- peaks. It was a gift of Karen Demby, School of Pharmacy, University of North Carolina at Chapel Hill. A VG 12-250 LC/MS (VG Masslab, Altrincham, UK; quadrupole MS) was used in the TSP mode: vaporizer temperature, 390 "C; desolvation chamber temperature, 270 "C; source temperature, 200 OC; VG "focus 4" electrode, 300 V. A Gilson HPLC system consisting of two Model 302 pumps, a Model 811 dynamic mixer, and a Model 802B manometric module (Gilson Medical Electronics, Middleton, WI) was used. Samples were injected into a Rheodyne, Inc. (Cotati, CA), Model 7010 injector fitted with a 50-rL loop. Water and methanol were HPLC grade (Fisher Scientific Co., Fair Lawn, NJ). Sources of other compounds were as follows: trifluoroacetic acid, Fairfield Chemical Co., Blythewood, SC; trichloroaceticacid and ammonium acetate, Fisher; hydrochloric acid, J. T. Baker Chemical Co., Phillipsburg, NJ. The solutions used were prepared from equal volumes of methanol and 0.1 M aqueous ammonium acetak, methanol, 0.1 M aqueous ammonium acetate, and 0.1 M aqueous trifluoroacetic acid; methanol, 0.1 M ammonium acetate, and 0.1 M trichloroacetic acid; and methanol, 0.1 M ammonium acetate, and 0.1 M hydrochloric acid.
+
+
and 8.3 respectively (18)and do not transfer protons to acetate ions (pK, of acetic acid = 4.75) in solution. However, in the gas phase they can be predicted to transfer a proton to acetate, according to the following argument using Table I. They may be considered either as imides (gas-phase acidity 1452-1443 kJ mol-') or as phenols with electron-withdrawing N (gasphase acidity ((1464 kJ mol-'), and are, even by this quick approximation, stronger acids than acetic acid; hence acetate should deprotonate them. Again, this is what happens (Table 111).
+
RESULTS AND DISCUSSION Few of the following analytes are appreciably ionized in the pH 7 solution used as mobile phase. Ions formed from them must result from an ionization process in the mass spectrometer source. It has been shown (3) that neutral molecules can be ionized in TSP LC/MS by proton transfer if the acid/base equilibrium between neutral analyte and additive ion reverses on going from condensed to gaseous phase, but that they are not ionized by proton transfer if the equilibrium is not reversed. Table I contains sufficient information to predict the position of equilibrium for most neutral organics likely to be eluted by (partially) aqueous solvents. Gaseous Acids Weaker Than Acetic Acid. T o rationalize the negative ion reactivity of the analytes, we estimate their gaseous acidity as follows. The steroids I and I1 have different alcohol functions, with gas-phase acidities estimated from Table I to be between 1573 and 1544 kJ mol-l, and a ketone function with a gas-phase acidity estimated to be 1530 kJ mol-l. (The solution pK,'s of alcohols and ketone functions are, respectively, 18 and 19 (13,and steroid molecules do not transfer protons to acetate ions (pK, of acetic acid = 4.75) in solution.) Gaseous acetate should not deprotonate these, for the gas-phase acidity of acetic acid is only 1458 kJ mol-', and clusters with acetate should form instead. This is, in fact, what happens (Table 111).
111
IV
\
VI
VI1
\
IX
VI11
Controlling the Appearance of the Mass Spectrum of the Analyte by Choice of Ionic Additive. Chloramphenicol propionate, X, contains a secondary benzylic alcohol and a
I
I
V
Acids of Strength Commensurate with That of Acetic Acid. The pKa's of the stilbestrol analogues VI-IX are unknown but should be similar to those of 4-ethylphenol (pK, = 10.18 (19))or 4-(l-propenyl)phenol (pK, = 9.80 (20));VI-IX cannot transfer protons to acetate ions (pK, of acetic acid = 4.75) in solution. However, their acidities can be estimated from Table I to be very close to that of acetic acid in the gaseous phase. Their spectra are given in Figure 1. Hexestrol, VI, has p-alkylphenol groups, with a gas-phase acidity estimated to be slightly greater than that of phenol, or about 1460 kJ mol-l; the gas-phase acidities of E-diethylstilbestrol, VII, E$-dienestrol, VIII, and indenestrol A, IX, would be roughly similar. These are all close to the gas-phase acidity of acetic acid, 1458 kJ mol-l. Thus, the case for deprotonation is indeterminate. Those acetate ions with a few kilojoules per mole excess internal energy can deprotonate molecules of VI-IX, and conversely molecules of VI-IX with a few kilojoules per mole excess internal energy cannot be deprotonated by acetate and form clusters instead. This participation in both processes is typical behavior in ion/molecule reactions for nearly thermoneutral reactions.
CH,OH
CH20H
&ZH
1663
OH NHCOCHCI,
O
?
N / OCH-CH-CH20COCH2CH, X
0
I
I1
Gaseous Acids Stronger Than Acetic Acid. The metribuzin metabolites 111, IV, and V, have pK,'s of 7.3, 10.0,
dichloroacetamide of a secondary amine, functional groups with solution pK,'s of about 18 (17) and about 12 (estimated from data for CH,COOH, Cl,CHCOOH, and CHBCONHz(21), respectively), that cannot donate a proton to acetate (pK, of acetic acid = 4.75). From Table I, we would assign the alcohol
1664
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
Table 111. TSP LC/MS Negative-Ion Mass Spectra with Ammonium Acetate Additive compound
I, hydrocortisone 11, corticosterone 111, DK (diketo metribuzin) IV, DA (deaminated metribuzin) V, DA DK (deaminated d i k e t o metribuzin) VI, hexestrol VII, E-diethylstilbestrol VIII, E$-dienestrol IX, indenestrol A X, chloramphenicol propionate@ w i t h trifluoroacetate with trichloroacetate
m/z (relative intensity) [M - 11[M + A]-
421 (100) 405 (100) 183 (100) 198 (100)
+
168 (100) 269 (15) 267 (25) 265 (60) 265 (100) 377 (50)
CONCLUSIONS From the values given in Table I, it should be possible to estimate gaseous acidities of many kinds of molecules typically analyzed by TSP LC/MS (or any other process dependent on gas-phase chemical ionization) so that the nature of their negative-ion spectra can be predicted from comparison with the gas-phase properties of other anions present, using the examples above as paradigms. Then, whether the peak at highest mass is the [M - 11- peak or the [M A]- peak can be controlled by suitable choice of A- as an additive. Similarly, the question of identification of an unknown peak as [M 11- or [M+ A]- can be addressed, Practically all organic compounds with ionizable hyrogen can be made to form easily identifiable [M + A]- ions (for example, by using trichloroacetate as a postcolumn additive (22));and the formation of [M- 11- or [M + A]- with different additives will provide information on the functionality of the unknown.
329 (100) 327 (100) 325 (100) 325 (33) 437 (35), 413 491 (100) 541 (loo), 413
+
M a j o r i o n of each cluster reported. [M Cl]-; C1- presumably available f r o m pyrolysis of analyte in some r u n s (Dougherty, R. C.; Roberts, J. D.; Biros, F.J. Anal. Chem. 1975,47, 54-59).
function a gaseous acidity of 1535-1525 kJ mol-' and the amide a gaseous acidity of 1480-1450 kJ mol-l. These are arrived at by assuming that substituent effects on 0 and N are similar, and that the gaseous acidity of dichloroacetic acid, which is not known, can be roughly estimated from a 10 kJ mol-' correction to two-thirds of the difference between CH,COOH and CF,COOH, the only appropriate acids whose acidities are known. Hence, in the gas phase the most acidic hydrogen in the compound is the amide hydrogen, and it is of the same order of acidity as acetic acid. From the previous examples, the prediction would follow that acetate will both deprotonate and complex with chloramphenicol propionate in the gas phase. On the other hand, in the other eluant mixtures, eq 2 proceeds favorably to the right for HA = trifluoroacetic acid, trichloroacetic acid, and hydrochloric acid, in the gas phase. Since trifluoroacetate, trichloroacetate, and chloride are far too weak to deprotonate chloroamphenicol propionate, they will form adducts only. As predicted, trifluoroacetate, trichloroacetate, and chloride form adducts with X (Table 111). Acetate both deprotonates and forms an adduct with it. Again, as with VI-IX, this dual reactivity is the behavior expected for exchange between two acids nearly equal in acidity, and the existence of a [M - 11peak confirms that the gaseous acidity of chloramphenicol propionate is greater than that of acetic acid, at least for some "hot" molecules. The results are consistent with a gaseous acidity of X comparable to that of acetic acid, 1458 kJ mol-', within the range estimated before.
LITERATURE CITED (1) Fenselau, C. I n "Ion Formaflon from Organlc SoMs; Benninghoven, A., Ed.; Springer-Verlag: Berlin, 1983; pp 90-100. (2) Garteiz, D. A.; Vestal, M. L. LC Mag. 1985, 3 , 334-346. (3) Bursey, M. M.; Parker, C. E.; Smith, R. W.; Gaskell. S.J. Anal. Chem. 1985, 57, 2597-2599. (4) Alexander, A. J.; Kebarle, P. Anal. Chem. 1986, 58, 471-478. (5) Anonymous. Chem. Eng. News 1985, 63(20), 38. (6) Parker, C. E.; Bursey, M. M.; Smlth, R. W.; Gaskell, S.J. J. Chromafogr. 1985, 347, 61-74. (7) Larson, J. W.; McMahon, T. 8. J. Am. Chem. SOC. 1984, 106, 517-521. (8) Larson, J. W.; McMahon, T. E. J. Am. Chem. SOC. 1983, 705, 2944-2950. (9) Clair, R. L.; McMahon, T. B. Can. J. Chem. 1979, 5 7 , 473-479. (10) Caldwell, G.; Rozeboom, M. D.; Kiplinger, J. P.: Bartmess, J. E. J. Am. Chem. SOC. 1984, 106, 4460-4667. (1 1) Bartmess, J. E. J. Phys. Chem. Ref. Data, in press. (12) Bartmess, J. E.; Mclver, R. T., Jr. I n Gas-Phase Ion Chemistry, Bowers, M. T., Ed.; Academic Press: New York, 1979; Vol. 2, pp 87-121. (13) Harrlson, A. G. Chemical Ionization Mass Spectrometry; CRC: Boca Raton, FL, 1983; p 36. (14) Squlres, R. R.; Bierbaum, V. M.; Grabowski, J. J.; DePuy, C. H. J. Am. Chem. SOC. 1983, 105, 5185-5192. (15) Fenselau, C.; Liberato, D. J.; Yergey, J. A,; Cotter, R. J.; Yergey, A. L. Anal. Chem. 1984, 56, 2759-2762. (16) Albro, P. W.; Parker, C. E.;Marbury, G. D.; Hernandez, 0.; Corbin, F. T. Appl. Specrrosc. 1984, 38, 556-562. (17) McEwen, W. K. J. Am. Chem. SOC. 1936, 5 8 , 1124-1129. Mester, T. C.; Hass, J. R.; (18) Albro, P. W.; Parker, C. E.; Abusteit. E. 0.; Sheldon, Y. S.; Corbin, F. T. J. Agr. FwdChem. 1984, 32,212-217. (19) van Hooidink, C.; Ginjaar, L. R e d . Trav. Chim. Pays-Bas 1962, 86, 449-457. (20) Lindberg, J. J.; Nordstrom, C. G.; LaurBn. R . Suom. Kemistil. B 1982, 35, 182-185. (21) Albert, A.; Serjeant, E. P. The Determination of Ionization Constants, 3rd ed.; Chapman & Hall: London, 1984; pp 138, 146. (22) Voyksner, R. D.; Bursey, J. T.; Pelllzzari, E. D. Anal. Chem. 1984, 5 6 , 1507-1514.
RECEIVED for review August 9,1985. Resubmitted November 12, 1985. Accepted February 24, 1986.
