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Anal. Chem. 1989, 6 1 , 1159-1164

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Atmospheric Pressure Spray Ionization for Liquid ChromatographyIMass Spectrometry Minoru Sakairi* and Hideki Kambara Advanced Research Laboratory, Hitachi Ltd., Kokubunji, Tokyo 185, J a p a n

Ionlzatlon characteristics and appilcablllty of the atmospherlc pressure spray (APS) method for liquid chromatography/mass spectrometry (LC/MS) are reported. I n thls system, small droplets containing cluster ions produced by a nebulizer at atmospheric pressure are introduced into a colilslon-induced dlssociatlon reglon to produce catlonlzed and/or protonated molecules. Thls method shows that the total positive ion lntenslty Is much hlgher than that of the negatlve ion lntenslty, wlthout or at low concentrations of ammonium acetate solutions. These asymmetric Ion formation characteristics are In contrast to the characteristics of thermospray Ionization using ammonium acetate soiutlons. The wide appilcabiiity of thls method for thermolabile and nonvolatlle compounds Is evidenced by the productlon of Intense catlonlred and/or protonated molecules for saccharides, peptldes, glycosldes, phosphoilplds, giycoilplds, and vitamin B,*. Fifty-picogram detection for sucrose Is also demonstrated.

INTRODUCTION Liquid chromatography/mass spectrometry (LC/MS) is an important area of analytical chemistry ( I ) . In LC/MS, ionization methods a t atmospheric pressure are of increasing importance (2-5). Previously, we developed a liquid chromatrography/atmospheric pressure ionization mass spectrometer (6-8),which has since been applied in various fields (9-11). In this atmospheric pressure ionization (API) method, LC effluents are vaporized by a nebulizer and a vaporizer, and vaporized sample molecules are ionized by a corona discharge using a needle electrode followed by ion/molecule reactions. Another widely used LC/MS system, developed by Vestal et al., is a thermospray (TSP) method (12,13). This is very sensitive to peptides and so on, and seems complementary to the API method. Since the API and TSP methods use a similar nebulizer consisting of a heated capillary, we investigated the possibility of TSP ionization a t atmospheric pressure, using the nebulizer and the vaporizer in the API ion source. In this experiment, the needle electrode for a corona discharge was demounted. However, this configuration did not produce any intense ions. T o investigate the reason for this, various ion source configurations and operating conditions were tested. Consequently, intense cationized and/or protonated molecules of nonvolatile compounds could be obtained when the vaporizer was demounted, the capillary head of the nebulizer was set close to an ion-sampling aperture, and the capillary was operated a t high temperature. The obtained mass spectra were quite different from those produced by the TSP method. We have called this technique an atmospheric pressure spray (APS) method. This paper reports the characteristics of this new ionization method and demonstrates the excellent ionization ability for nonvolatile and thermolabile compounds.

EXPERIMENTAL SECTION Apparatus. Schematic diagrams of the liquid chromatograph/atmospheric pressure spray mass spectrometer and the

liquid chromatograph/atmospheric pressure ionization mass spectrometer are shown in Figure 1, parts a and b, respectively. The APS ion source is very simple and require only a few modifications from the previously reported API ion source (8). These modifications mean only demounting the bored stainless steel block and the needle electrode and setting the capillary head close to the first electrode. LC. A Hitachi 655A high-performance liquid chromatograph (HPLC) with a loop injector and a Hitachi L-5000 LC controller was used. A column (4 mm X 150 mm) packed with Hitachi gel No. 3056 (ODS) for a reversed-phasechromatography (RPC) mode was used for separating a mixture. The LC was connected with the nebulizer through a Teflon tube (1.6-mm o.d., 0.25-mm i.d., 1 m long) because a high voltage was applied to the nebulizer. Nebulizer. The nebulizer consisted of a stainless steel capillary (1.6-mm o.d., 0.1-mm i.d.) brazed to a stainless steel block (40 mm long), which could be heated to 550 "C with temperaturecontrolled cartridge heaters. This nebulizer structure was similar to that of the TSP method (12). The capillary head of the nebulizer was set 5 mm away from the aperture of the first electrode,and the capillary axis and f i t electrode axis were joined in alignment, in order to efficiently introduce nebulized small droplets to the intermediate region. Mass Spectrometer. Small droplets, including cluster ions produced by the nebulizer at atmospheric pressure, were introduced into the ion source housing and the mass analyzing region through the intermediate region, which was located between the first electrode (with a 250-fim aperture) and the second electrode (with a 400-gm aperture). A drift voltage was applied to the intermediate region to dissociate cluster ions into cationized and/or protonated molecules (8). When a voltage was applied to the intermediate region, cluster ions were accelerated and collided with neutral molecules in the intermediate region. As a result, the kinetic energy of the cluster ions was partly converted into the internal energy of the cluster ions. Cluster bonds are dissociated if the internal energy of the cluster ions is sufficient. Thii collision-induced dissociation (CID) technique was very useful for breaking cluster ions. Normally the drift voltage was set to 100 V. In this case, the first and second electrode potentials were 3.1 and 3 kV, respectively. Ion acceleration energy was determined to be 3 keV by the second electrode potential. The potentials of the nebulizer and the first electrode were the same. The intermediate region was evacuated with a 1500 L/min mechanical booster pump and a 500 L/min rotary pump to about 60 Pa. A discharge suppressor was installed inside a polymer hose connecting the intermediate region with the mechanical booster pump. A cold trap to prevent the excess accumulation of solvents in the pump oil was unnecessary. This was very convenient for long and/or continuous operations. The ion source housing and the mass analyzing region were evacuated with two 700 L/s diffusion pumps to about 1 x and 1 x IO4 Pa, respectively. A Hitachi M-80B double-focusing mass spectrometer was used, and a Hitachi 0101 data acquisition system was used for obtaining LC/MS data. The scan range was 1-1500 m u , and the scan speed was 1500 amu/4 s under typical measurement conditions. Operation. The temperature of the stainless steel block in the nebulizer and the drift voltage in the intermediate region were set at 480 " C and 100 V, respectively, because a maximum intensity of the cationized molecule for sucrose ( m / z = 365 for M + Na)+)could be obtained under these conditions. Intense cationized and/or protonated molecules for various kinds of nonvolatile compounds could also be observed under these conditions. In the following experiments,the temperature of the stainless steel

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Flgure 1. Schematic diagrams of (a) the APS ion source and (b) the API ion source: (1) IiquM chromatograph, (2)stainless steel capillary, (3) stainless steel block, (4) first electrode with aperture, (5) second electrode with aperture, (6) tens, (7) ceramic plate, (8) intermediate region, (9)bored stainless steel block, (10)needle electrode for corona

Flgure 2. Variation of positive and negative ion intensities of 100% water and 0.5 M ammonium acetate solution with the temperature of

the stainless steel block in the nebulizer.

PA. This value corresponded closely to the total positive ion currents monitored a t the total ion monitor, with the ion transmission efficiency through the two apertures taken into discharge. consideration. T h i s was because the negative ion currents were negligible. The ion currents increased monotonically with the block and the drift voltage were set to these levels. temperature to 480 "C and then decreased slightly. It has been Mass spectra of nonvolatile compounds were measured by flow reported that the optimum temperature with the use of an injection analysis without use of a column, using 5 pg of each indirect heating nebulizer a t a flow rate of 1mL/min is about sample. One hundred percent distilled water was used for sac250 "C and that the disappearance of ion intensities on the charides and glycosides, and water/methanol (50/50) was used high-temperature side is observed in TSP ionization (12). As for peptides and phospholipids as mobile phases, at a flow rate the liquid phase is converted to the gase phase by heating the of 1mL/min. For glycolipids, methanol/chloroform (70/30) was capillary to a high temperature, the disappearance of the ion used at a flow rate of 1mL/min. Analysis of a saccharide mixture was conducted with the RPC type column and a mobile phase current is thought to be caused by a recombination of positive of water at a flow rate of 1 mL/min. and negative ions produced in the gas phase in the stainless Samples and Reagents. Xylose, sucrose, lactose, raffinose, steel capillary and the neutralization of ions resulting from stachyose, a-cyclodextrin, P-cyclodextrin, y-cyclodextrin, ditheir collision with the capillary's inner wall (14). In APS myristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, ionization, clear current cessation was not observed in the distearoylphosphatidylcholine, digitoxin, digitonin, and vitamin stainless steel block temperature region from 300 t o 540 "C, B12were purchased from Sigma Chemical Co. (St. Louis, MO). [Leu5]Enkephalin,[Met5]enkephalin, [~-Ala~,Met~]enkephalin, although the total ion intensity in distilled water tended to decrease above the nebulizer temperature of 480 "C. The proctolin, eledoisin related peptide, Des-Argg-[Leug]bradykinin, temperature where ion currents decrease drastically angiotensin 111, [Sarl,Iles]angiotensin 11, [ A s ~ ~ ~ ~ , A r ~ ] v a s o t o c i critical n, bradykinin potentiator C, bradykinin, luteinizing hormone reis thought to correlate well with the gas pressure in the caleasing hormone, angiotensin I, Met-Lys-bradykinin, [Tyr-g] pillary head region. Thus, the lack of cessation in APS ionsubstance P, and BAM-12P were purchased from the Institute ization seems to be the reason that the critical temperature of Peptides (Osaka,Japan). Lactosyl ceramide and globoside were for disappearance of the current is higher than that for TSP kindly supplied by Mitsubishi-Kasei Institute of Life Sciences ionization a t a low pressure. The jet temperature in APS (Tokyo, Japan), and a saccharide mixture from soybeans was ionization monitored by a thermocouple was about 300 "C kindly supplied by Nissei Sangyo (Tokyo,Japan). Distilled water, when the stainless steel block temperature was 480 "C. This methanol, and chloroform of LC grade were purchased from Wako temperature was also much higher than that of TSP ionization Chemical Co. (Tokyo, Japan). (150-200 " C ) (12). RESULTS AND DISCUSSION The primary charging mechanism of TSP ionization using APS Ionization Characteristics. The APS ion source ammonium acetate solution as a mobile phase is explained structure was almost the same as that of the TSP method by the statistical symmetric charging mechanism described except that small droplets were produced by the nebulizer a t by Dodd (15). This idea is supported by the fact that maxatmospheric pressure and the temperature of the stainless steel imum ion currents are nearly the same for both positive and block in the nebulizer was much higher. However, the mass negative ions in TSP ionization (12). Figure 3 shows the spectra, in which intense cationized molecules such as (M + maximum total ion intensities of positive and negative ions Na)+ and (M + K)+ were observed, were very different from as a function of ammonium acetate concentration in APS those obtained by the TSP method. Additionally, observation ionization. The difference between APS and TSP ionizations of these cationized molecules did not require adding alkalican be clearly seen. At high ammonium acetate concentrametal salts to solutions. tions, positive and negative ion intensities are close, which Figure 2 shows the variation of positive and negative total corresponds well to the result obtained by TSP ionization. ion intensities of distilled water and 0.5 M ammonium acetate However, a t low concentrations of ammonium acetate or solution with the stainless steel block temperature in the without ammonium acetate, the ion intensity of the positive nebulizer. This level of ammonium acetate solution is usually ions is much higher than that of the negative ions. For exused in thermospray ionization. The total ion intensities were ample, the ratio of positive to negative ion intensity for dismonitored with the total ion monitor located in the ion source tilled water is about 20 to 1. In addition, the observed ion housing. The total ion intensity a t atmospheric pressure of species were mostly cationized molecules, which differed from distilled water just after being nebulized a t 480 "C was 1.5 those observed in TSP ionization. Even if a large amount of'

ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989

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ammonium acetate was added and the estimated concentrations of Na+ (1 X M) and NH4+ (0.5 M) were quite different from each other, the observed Na+ and NH4+intensities were almost comparable. This suggests that the ion formation in APS is greatly affected by the chemical property of the ionized species. These features of APS ionization cannot be explained only by the symmetric charging mechanism as discussed in TSP ionization. Loeb reported that static electrification might involve two or more of the various basic mechanisms such as the spray electrification mechanism that included symmetric or asymmetric charging, the contact potential mechanism, the electrolytic mechanism, and the frictional electrical mechanism (16). It seems that the asymmetric behaviors between positive and negative ions and between NH4+ and Na+ reveal the possibility of other charging mechanisms such as contact or Volta electrification, asymmetric electrification, and so on. In conclusion, in APS ionization, a more complicated charging mechanism must be considered than the symmetric charging mechanism. Figure 4 shows the APS mass spectra of sucrose obtained with the use of water and 0.1 M ammonium acetate solution. The observed main background ions for the water mobile phase were Na+ ( m / z = 23), K+ ( m / z = 39), and H30+ ( m / z = 19). These were produced from the dissociation of cluster ions. The sucrose mass spectrum obtained with water as the mobile phase provides (M + Na)+ ( m / z = 365) and (M + K)+ ( m / z = 381) as ions relating to samples. By addition of ammonium acetate, NH4+ ( m / z = 18), CH3COONH4+( m / z = 7 8 ) ,and (M NH4)+ ( m / z = 360) could also be observed. Even in 0.1 M ammonium acetate solution, (M + Na)+ is more abundant than (M NH4)+. Additionally, the ratio of (M + Na)+ to the sum of (M Na)+ and Na+ was almost the same, in spite of the addition of ammonium acetate. On the other hand, the APS mass spectra of [~-Ala~,Met~]enkephalin obtained by using water and 0.1 M ammonium acetate solution as mobile phases are shown in Figure 5. The spectrum obtained with water provides (M H)+ ( m / z = 588), (M + Na)+ ( m / z = 610), and (M K)+ ( m / z = 626), and by addition of ammonium acetate, (M H)+ increases while (M + Na)+ and (M K)+ decrease. This indicates that addition of salts increases protonated molecule formation and negatively affects cationized molecule formation. Of course, many positive and negative ions might be produced in a spray, but it seems that most of them recombine soon after nebulization and are insignificant to ion currents monitored at the total ion monitor. Application. Applicability. To evaluate the applicability of APS ionization to ionizing thermolabile and nonvolatile compounds, full mass spectra of several compounds were measured by flow injection analysis without use of a column. The stainless steel block temperature of the nebulizer was 480

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"C, and the drift voltage was set at 100 V. Table I shows major observed ions, and Figure 6 shows the APS mass spectrum of globoside, which is a glycolipid, as one example. As shown in Table I, the APS ion source produces intense cationized and/or protonated molecules for saccharides, peptides, glycosides, phospholipids, glycolipids, and vitamin BIZ. It is noteworthy that intense (M + Na)+ and (M + K)+ of saccharides can be observed without using buffers (17-19). It is also an advantage that the intensities of multiply charged ions, which often cause confusion, are weak. The mass spectra of luteinizing hormone releasing hormone (LHRH) in the APS

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Table 1. Major Observed Ion Species of Nonvolatile Compounds and Their Relative Intensities mobile phase (1 mL/min)

compds

xylose C5H1005 (150) glucose C6Hlz06(180) sucrose C1zHzzO1l(342)

maltose C12H22011 (342) raffinose C18H32016 (504) stachyose C24H42021 (666) maltopentaose C3OH5zOz6 (828) a-cyclodextrin C36H&30 (972) P-cyclodextrin C42H70035 (1134) y-cyclodextrin C48&,040(1296) [Leu'lenkephalin C28H37N507 (555) [Met'lenkephalin C27H35N507S (573) [~-Ala~,Met~]enkephalin Cp8H4,N5OIlS(587) proctolin C30H48N808 (648) eledoisin related peptide C34HB8N8011S (706) Des-Argg-[Leu8]bradykininC41H77N11017 (869) angiotensin 111 C46H&"209 (930) (697) [Sar',Iles]angiotensin 11 C46Hg7N13017 [A~u~~~,Arg~]vasotocin C46HBSN14020 (998) bradykinin potentiator C C51H97N11023 (1051) bradykinin CSOH73N15011(1059) luteinizing hormone releasing hormone C55H93N17022 (1181) angiotensin I C6zH8gN,7014 (1295) Met-Lys-bradykinin C61H94N18013S (1318) [Tyr-81 substance P C63Hg8N18014S (1362) BAM-12P digitoxin C41H&3

(764) digitonin C6BH92029 (1228)

dimyristoylphosphatidylcholine C36H72N08P(677) dipalmitoylphosphatidylcholine C38H76NOBP (733) diastearoylphosphatidylcholineC4,H,N08P (789) lactosyl ceramide C46H87N013 (861)

globoside C6gH130N2022 (1338) (1354) vitamin B12Cs3HB8CoN14P

8

1088

688

HZO HZO H2O H2O H20 HZO HZO H2O HZ0 HZO HzO/CH3OH (50/50) HZO/CH,OH (50/50) HZO/CH,OH (50/50) HZO/CH3OH (50/50) HzO/CH3OH (50/50) HZO/CH30H (50/50) HzO/CH30H (50/50) HzO/CH3OH (50/50) HzO/CHsOH (50/50) HZO/CH30H (50/50) HzO/CH30H (50/50) HZO/CH,OH (50/50) HzO/CH30H (50/50) HZO/CH3OH (50/50) HzO/CH30H (50/50) HzO/CH3OH (50/50) HvO Hi0 HzO/CH3OH (50/50) HZO/CH,OH (50/50) HZO/CH3OH (50/50) CH3OH/CHCl, (70/30) CH,OH/CHCl, (70/30) HzO/CHsOH (50/50)

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Flgure 6. Globoside mass spectrum.

and TSP methods clearly show the contrast of these multiply charged ions (19). Lipids, which can be dissolved only in solutions extremely high in organic content, generate abundant cationized molecules in APS ionization without external ion sources, while external ionization is frequently required in other LC/MS systems (20, 21). Table I also shows an interesting feature of APS ionization. Compounds consisting of nitrogen atoms produce abundant (M + H)+ in addition to (M + Na)+ and (M + K)+. However, except for digitonin, only (M + Na)+ and (M + K)+ are observed in compounds with oxygen atoms but no nitrogen atoms. Therefore, it is reasonable to conclude that hard acids of alkali metals and hard bases of oxygen atoms are held together, and protons bind to nitrogen atoms with high proton affinities in the observed protonated molecules. The APS mass spectrum of globoside (MW 1338) shown in Figure 6 clearly reveals characteristics of APS ionization. Abudant cationized molecules of (M + Na)+ ( m / z = 1361) and (M + K)+ ( m / z = 1377) are observed with few fragment ions.

major obsd ions and re1 intens, % (M + H)+ (M + Na)+ (M + K)+ other ions

556 (14) 574 (8) 588 (24) 649 (100) 707 (36) 870 (92) 931 (100) 968 (84) 999 (100) 1052 (5) 1060 (100) 1182 (100) 1296 (100) 1319 (100) 1363 (100) 1423 (100) 1229 (16) 678 (15) 734 (10) 790 (10) 1355 (60)

173 (100) 203 (100) 365 (100) 365 (100) 527 (100) 689 (100) 851 (100) 995 (100) 1157 (100) 1319 (100) 578 (100) 595 (100) 610 (100) 671 (58) 729 (100) 892 (100) 953 (25) 990 (100) 1021 (32) 1074 (74) 1082 (23) 1204 (22) 1318 (228) 1341 (27) 1385 (81) 1445 (28) 787 (100) 1251 (100) 700 (100) 756 (100) 812 (100) 884 (100) 1361 (100) 1377 (100)

381 (26) 381 (18) 543 (18) 705 (12) 861 (21) 1011 (10) 1173 (14) 1235 (12) 594 (21) 612 (10) 626 (28) 687 (9) 745 (26) 908 (15) 969 (14) 1006 (18) 1090 (68) 1095 (11) 1334 (12) 1357 (10) 1401 (43) 1461 (19) 803 (5) 1267 (23) 716 (20) 772 (22) 828 (15) 900 (22) 1377 (39)

245 (21)

632 (18)

1096 (100)

495 (16) 551 (18) 607 (15) 1329 (30)

The ions at m / z = 1359 and 1375 are due to impurities with unsaturated hydrocarbons of ceramides. Although glycolipids such as globoside have gained attention in mass spectrometry in recent years, it has been difficult to observe intense molecular ion species with LC/MS. The APS method is very promising for analyses of glycolipids, because intense cationized molecules can be observed. Structure Elucidation. As demonstrated above, the common characteristic of mass spectra produced by the APS method is the low abundance or absence of fragment ions. However, reduced fragmentation provides insufficient structural information for identifying unknown compounds. One approach to overcoming this difficulty is the MS/MS method reported by Henion et al. ( I ) . However, this method is very expensive and hard to operate, although it is well established. Therefore, a CID technique is applied in the intermediate region to increase fragmentation as well as to break cluster ions in the liquid chromatograph/atmospheric pressure spray mass spectrometer. This method is very useful because a mass spectrometer can record alternate high and low collision energy spectra and, therefore, easily generate alternate high and low fragmentation mass spectra. Figure 7 shows APS mass spectra of digitonin, which is a glycoside, at drift voltages of 100 and 200 V. Although significant fragment ions are not observed in the mass spectrum at 100 V, many are observed at 200 V. The ion at m/z = 133 corresponds to the pentose moiety and that at m / z = 163 to the hexose moiety. These ions are useful for showing the existence of pentose and hexose in a measured compound. The ions due to the loss of pentose and hexose, accompanied by protonation, are observed at m / z = 1097 and 1067, respectively. The presence of a hexose-hexose structure is shown by the ion at m / z = 325. The ions corresponding to the digitogenin moiety are shown as the ions at m / z = 449

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Table 11. Detection Limits Obtained with a SIM Mode in APS Ionization compds

detected ions

mobile phase (1 mL/min)

detection lim, pg ( S I N = 10)

sucrose C12H22011 (342) raffinose C18H32016(504) proctolin C3,,HaN808(648) eledoisin related compd C34H88N8011S (706)

365 (M + Na)+ 527 (M + Na)+ 649 (M + H)+ 729 (M + Na)+

HzO HzO H20/CH30H(50/50) H20/CHBOH(50/50)

30 75 160 50

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by protonation and m / z = 471 by sodiation, while the residual moiety is shown as the ions at m / z = 781 and m / z = 803 by sodiation. The ions a t m / z = 611 and 633 correspond to the hexose-digitogenin structure accompanied by protonation and sodiation, respectively. As demonstrated here, significant fragment ions, which are very useful for structure elucidation, can easily be obtained by a CID technique a t a high drift voltage. Mixture Analysis. Mixture analyses are very important for investigating components contained in samples. As an example, a saccharide mixture produced from soybeans, in which sucrose was a main product, was measured. This measurement was performed under normal operating conditions (drift voltage of 100 V) so that just enough collision energy was used to break the cluster ions without fragmenting the sample ions. The obtained mass chromatograms (mlz = 203,365,527,689, and 851) are shown in Figure 8. Although separation by a HPLC is not good, these chromatograms indicate that the sample includes several components. If the main product of the sample is known to be sucrose and the APS mass spectra show few fragment ions, it can easily be estimated that the ions at m / z = 203,527, and 689 correspond to glucose (or fructose), raffinose, and stachyose, respectively. The quantitative analysis for sucrose could be performed because the relationship between the cationized molecule intensity and the amount of sucrose injected showed good

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Mass chromatogram of a 50-pg sample of sucrose, obtained by a SIM mode. Figure 9.

linearity in the range between 10 ng and 10 pg for a cyclic scan mode (scan range: 100-500 amu). Sensitivity. The sensitivity of APS ionization for cationized or protonated molecules is dependent on the type of samples. The detection limits (SIN = 10) by a selected ion monitoring (SIM) mode without use of a column were estimated from the results of 50-pg injections for sucrose and eledoisin related compounds and 200-pg injections for raffinose and proctolin. The values shown in Table I1 are averages of the detection limits obtained from three times injections for each sample. These results show the high sensitivity of APS ionization. The mass chromatogram of sucrose obtained by a SIM mode (mlz = 365 for (M Na)+) with a 50-pg sample is shown in Figure 9. In addition, from the chromatogram obtained, any con-

+

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tribution to band broadening by the nebulizer is almost unalthough a little peak is Observed' Regu1ar oscillations in the ion intensity, which occur with a period of about 6 s, are due to flow fluctuation introduced by the HPLC pump.

(9) Handa, S.; Kushi, Y. Satellite Symposium of the ISS-ASN Joint Meeting, Pueruto La Cruz. Venezuela, 1987. (10) Kozuka, M,; Hashimoto, K,; Takasaki, K,; Konoshjma, K,; Kat&, y,; Amano, T. Poster Abstr. J p n . - U S . Congr. Pharm. Sci. 1987, 205. (11) Sakairi, M.; Kambara, H. Anal. Sci. 1988, 4 , 141. (12) Blakley. C. R.; Vestal, M. L. Anal. Chem. 1983, 5 5 , 750. (13) . . Liberato. J. D.: Fenselau. C. C.: Vestal. M. L.: Yeraev. A. L. Anal. Chem. 1983. 55, 1741. (14) Vestal, M. L.; Fergusson, G. J. Anal. Chem. 1985. 5 7 , 2373. (15) Dodd, E. E. J . Appl. Phys. 1953, 224, 73. (16) Loeb, L. B. Science 1945, 102, 1363. (17) Hsu, F. F.; Edmonds. C. G.; McCloskey, J. A. Anal. Left 1986, 19, 1259. (18) Butfering, L.; Schmelzeisen-Redeker. G.; Rollgen, F. W. J . Chromatog. 1987, 394, 109. (19) Robins, R. H.; Crow, F. W. Presented at the 34th ASMS Conference on Mass Spectrometry and Allied Topics, Cincinnati, OH, 1986. 570. (20) Kim. H. Y.; Salem, N., Jr. Anal. Chem. 1986. 58, 9. (21) Kim, H. Y.; Salem, N., Jr. Anal. Chem. 1987, 5 9 , 722. 1

LITERATURE CITED Covey, T. R.; Lee, E. D.; Bruins, A. P.; Henion, J. D. Anal. Chem. 1988, 58, 1451A. Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 5 9 , 2642. Covey, T. R.; Bruins, A. P.; Henion, J. D. Org. Mass. Spectrom. 1988, 23, 178. Whitehouse, c, M,; D ~ R , N,; ~ yamashita, ~ ~ M.; F~ ~ J,, B, ~ Anal, ~ , Chem. 1985, 5 7 , 675. Thomson, B. A.; Iribarne, J. V.; Dziedzic, P. J. Anal. Chem. 1982, 5 4 , 2219. Sakairi. M.; Kambara, H. Mass Spectrosc. 1983, 31,87. Sakairi, M.; Kambara. H. Presented at the 35th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO, 1987. 407. Sakairi, M.; Kambara, H. Anal. Chem. 1988, 6 0 , 774.

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RECEIVED for review October 4,1988. Accepted February 13, 1989.

CORRESPONDENCE

Absorption Spectroscopy Sir: Considerable progress has been made over the last decade toward the development of infrared spectroscopy as a probe of the vibrational and structural properties of species adsorbed on electrode surfaces. Fourier transform infrared reflection absorption spectroscopy (FTIRRAS) has become perhaps the most versatile technique for acquiring in situ data, as evidenced by the growing number of applications reported in the literature (1,2). Particular attention has been focused on the use of this methodology for the study of interactions involving simple ions and metal surfaces. In the case of Pt in sulfuric acid solutions, spectral features associated with both sulfate and bisulfate were observed in the p-polarized spectra (3). The relative intensities and frequencies of these bands were found to be a function of the applied potential, the latter providing strong evidence that the signals are indeed associated with species adsorbed on the electrode surface. The occurrence of electrolyte bands in potential difference FTIR spectra, however, may not always be ascribed to species adsorbed on the electrode surface. This is due to the fact that the thickness of the electrolyte layer between the electrode and the window is most often smaller than the wavelength of infrared radiation ( 4 ) . Hence, all IR-active species present in the thin layer can be detected with radiation polarized parallel to the plane of incidence, a factor that must be taken into account in the interpretation of spectral data. This communication will present in situ FTIRRAS spectra for Au and Pt electrodes in nitric and perchloric acid solutions and electrolytes containing both perchlorate and nitrate ions. The results obtained have afforded rather conclusive evidence that the absorption bands observed for potentials in the oxide formation region can be attributed to the relatively large amount of charge passed between the working electrode in the thin-layer cell and the externally located counter electrode, resulting in the migration of anions into the gap of the thinlayer cell. Specifically, the charge of the protons produced during anodic film formation must be compensated by the entrance of anions into the gap. 0003-2700/89/036 1-1164$01.50/0

EXPERIMENTAL SECTION The electrochemical cell involved in these studies is shown in Figure 1A. The Au electrode, a cylinder of 19-mm diameter and 2 mm-height, was cast in a Kel-F block exposing a circular area of about 2.8 cm2. It was then polished with a series of alumina powders of successively smaller size down to 0.05 pm, washed with nitric acid, and finally cleaned under ultrasonic agitation in ultrapure water. The electrode holder consisted of a threaded Teflon cap that could be screwed onto the back of the Kel-F piece so as to trap loosely a Teflon rod ending in a hemispherical head of larger diameter. After the cell was assembled, the Teflon rod, which was inserted in the back of the cell body, could be pushed against a hemispherical cavity machined in the back of the Kel-F block, forcing the electrode surface parallel to the CaF, window. Prior to the optical measurements, the electrode was moved away from the window. It was then subjected to a series of oxidation-reduction cycles between potentials close to hydrogen evolution and oxygen generation until voltammetry curves characteristic of clean Au were obtained. The electrolyte was then exchanged and the electrode pushed against the window for a complete series of spectroscopic measurements. Infrared spectra were obtained with a Michelson-Genzel type FTIR instrument (IR/98, IBM Instruments, Inc.) equipped with a liquid nitrogen cooled HgCdTe detector. The optical attachment involved in the in situ measurements is shown schematically in Figure 1B. Reflection spectra at a given potential were obtained by adding 500 interferometric scans. The data are presented in the form of -A.R/R vs wavenumber, where AR = (RmPh - R ) and R is the reflection spectrum at an arbitrary reference potential. Ultrahigh-purityHNO, and HCIOI were purchased from Baker and the solutions made with ultrapurified water obtained from a modified Gilmont distillation system. All potentials were measured versus a standard calomel electrodeplaced in an external compartment connected to the cell through a long Teflon capillary. A Au wire was used as a counter electrode. All measurements were conducted at room temperature. The experiments with Pt electrodes (0.79 cm2jwere carried out with a commercially available spectroelectrochemicalcell designed specifically for in situ IR measurements (Chemical Electronics Associates, Hickory, NCj. 1989 American Chemical Society