Anal. Chem. 1992, 6 4 , 953-957
chemical species. Despite the higher noise level, the spectra of the pure components (Figure 9) were obtained completely similar to those obtained for the systems with chromatographic resolution of 0.5 (Figure 6). CONCLUSION We have shown that the new method called heuristic evolving latent projections (HELP) is superior to some of the most commonly methods of factor analysis for resolution of mixtures where minor chemical species coelute with major ones. The crucial factors explaining the excellent results are: (i) the use of the zero-component region in order to establish a realistic criterion for detection of species and thus the number of chemical species in a mixture, (ii) the detection and use of the selective information using latent-projective graphs and local rank analysis, and (iii) the enhanced capability for detection and resolution provided by the method‘s ability to point at interesting local regions that deserve a more thorough investigation. The method‘s character of a datascope and a data-driven inductive procedure provide the analyst with a tool that enables rapid answers to the key points in evaluation of multicomponent data: (i) How many chemical species are there above detection limit? (ii) Where are the selective regions? (iii) Can the mixture be resolved or are new experiments needed? Finally, we would like to point out that the solution of the present problem does not suggest a lower limit for the resolution power of the HELP method with respect to concentration of chemical species, chromatographic resolution, or number of unresolved chemical species. Actually, work in progress shows that we can resolve a mixture of isomers where a minor component represents less than 1.0% of a major coeluting one.18 Furthermore, we have recently been able to
953
resolve six-component mixtures of coeluting chlorophyll degradation produds in the presence of large correlated noise and base-line offset.2 ACKNOWLEDGMENT Yi-zeng Liang is grateful for a postdoctoral fellowship from the Royal Norwegian Council for Scientific and Industrial Research (NTNF). REFERENCES (1) Keller, H. R.; Massart. D. L. Anal. Chlm. Acta 1881, 246, 279-290. (2) Llang. Y-2.; Kvalheim. 0. M.; Rahmani, A.; Brereton, R. G , J . Chemom., submltted for publicatlon.
(3) Wurrey, C. J.,; Gurka, D. F. I n ApplicerrOn of FT-IR spectroscopy; Durig, J. R., Ed.: Elsevler: Amsterdam, 1990. (4) Maeder. M.; Zuberbuehler, A. D. Anal. Chlm. Acta 1888, 181. 287-291. (5) Maeder. M. Anal. Chem. 1987, 59, 527-530. (6) Geladi, P.; Wold. S. che”.Intell. Lab. Syst. 1887. 2 , 273-281. (7) Kvalhelm, 0. M.; Llang, Y.-r. Anal. Chem.. preceding paper in thls
issue. (8) Kvalheim, 0. M. Chemom. Intell. Lab. Syst. 1887, 2 . 283-290. (9) Kvalheim, 0. M. che”.Intell. Lab. Sysf. 1888, 4 , 11-25. (10) Lewi, P. Ch/m& M e ~ ~ z I n1087, e Aprll. 18-28. (11) Golub, G. H.; Van Loan, C. F. Metrfx ComputamOns, 2nd ed.;The Johns Hopklns Unhrerslty Press: Baltimore, 1989. (12) Wold, S.; Esbensen, K.; Wadi, P. Chemom. Intel. Lab. Sysr.1987.
2, 37-52. (13) Malinowskl, E. R.; Howery, D. G. Factwanalysls in chemlsby; Wiley 8 Sons: New York, l9SO pp 59-100. (14) Rothman, L. D.; Crouch, S. R.; Ingle. J. D., Jr. Anal. Chem. 1976, 47, 1226-1233. (15) Ingle, J. D., Jr.; Crouch, S. R. Anal. Chem. 1872, 44. 1375-1386. (16) Lorber, A. Anal. Chem. 1888, 54, 1167-1172. (17) Kvalheim, 0. M.; Karstang, T. V. Chemom. Intel/. Lab. Sysf. 1987, 2, 235-237. (18) Keller. H. R.; Massart, D. L.; Llang, Y.-2.; Kvalheim, 0. M. Anal. chkn. Acta, submltted for publication.
RECEIVED for review August 8, 1991. Accepted January 2, 1992.
Ultraviolet Resonance Raman Spectroscopy of 4-Aminopyridine Adsorbed on Zeolite Y Michael R. Jakupca a n d P r a b i r K. Dutta*
Department of Chemistry, The Ohio State University, 120 West 18th Avenue, Columbus, Ohio 43210
Resonance Raman spectroscopy b used in this paper to examine the weak acid sites of zeolite surfaces. 4-Aminopyrldhe, a base wlth a pK, of 9-17, b used as a probe. Upon protonath by the zeolite, the pyridine's maJor UV electronk absorption band undergoes a bathochromic shift. By uslng excttatlon that cdncides wlth the maximum d thk abrorptbn band (264 m),it is possible to selectively study the Raman spectra of the protonated species and detect the presence of weakly acldic hydroxyl groups.
INTRODUCTION Zeolites are used as catalysts in a number of processes such as petroleum cracking, NO, reduction, isomerization, alkylation, and dealkylation rea~tions.l-~ In order to understand their role in these reactions, it is important to understand the
* Author to whom correspondence should be addressed. 0003-2700/92/0364-0953$03.00/0
nature, location, and strength of their acid sites. Much of the characterization has focused on the Bronsted acid sites located inside the cages of zeolites. Typical ways to study these sites have been by using infrared spectroscopy of basea such as NH3, pyridine, and quinoline adsorbed on acid sites, simple visible color changes of color indicators, and titrations of zeolite surfaces against amine solutions using Hammett indicators to calculate the endpoint.’r4+ Recently, our group has shown the advantages of resonance Raman spectroscopy using visible excitation in distinguishing between acid and base forms of dye molecules adsorbed on zeolites.’ Different dyes with varying pKis can be used to qualitatively classify the acid strengths of different sites. However, many of the dyes that have been used as color or Hammett indicators have radii which are larger than the cage openings in zeolites (typically 4-8 A). This may lead to size selectivity and may not accurately reflect true site acidity. Thus it would be advantageous to use small molecules which could freely move about in the inner surfaces of the zeolite. Since smaller molecules typically 0 1992 American Chemical Society
854
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
have absorption bands in the ultraviolet, these cannot be used as color indicators. Diffuse reflectance spectra of such adsorbed species are often very broad and do not provide specific structural information. In this study, we have chosen to develop the technique of UV resonance Raman spectroscopy to follow the transitions of conjugate acid/base molecules on zeolite Y. In order to illustrate the sensitivity and selectivity of UV resonance Raman spectroscopy, we have elected to study weakly acidic surface sites. These sites do not have any catalytic relevance per se, but serve as a useful model in developing the use of UV resonance Raman spectroscopy for such systems. In the framework of a zeolite, the individual Si atoms are bound to either other Si atoms or an A1 atom by bridging oxygen, (-Si(0)2-O-Si(O)2-) or (-Si(0)2-O-Al(O),-)-X+ (X = counterion such as Na+ or H+). Dehydrated samples of completely alkali-exchanged zeolites do not contain any framework Bronsted acid sites. However, a weak infrared band is still observed at approximately 3740 cm-' and can be ascribed to lattice-terminating silanol groups which are exposed on the external surface and at defects within the These groups are resistant to ion exchange and high-temperature heating. Since most of the zeolite's surface area is contained within its interior framework (approximately 99%), there are relatively few of these acidic sites. Coverages have been calculated at approximately lozoOH per cm3 for crystals 1 pm a c r o ~ s . ~ In this study we have chosen a small molecule, 4-aminopyridine, as a probe to study the weak acid sites of a zeolite Y. Because of ita size, this molecule is able to move freely throughout the zeolite framework. It is a reasonably strong base (pK, -9.17) and its interaction with the acidic groups of the zeolite is of interest. The absorption band of 4-aminopyridine undergoes a 20-nm red shift upon protonation, and ita conjugate acid has a large absorptivity at its absorption maximum, 264 nm, which is nearly coincident with the fourth harmonic of a Nd:YAG laser, 266.2 nm. These properties of the molecule make it an attractive model to evaluate the possibility of using UV resonance Raman spectroscopy to examine the presence of low levels of hydroxyl groups. W resonance Raman of biologically relevant systems has been extensively studied over the past decade.loa It has clearly been shown with solutions of systems such as nucleic acids, proteins, amino acids, and polycyclic aromatic hydrocarbons that excitation in the UV region provides improved sensitivity and selectivity.1° Increasing attention is being paid to the study of solids and interfaces.l& We have developed the methodology necessary for obtaining Raman spectra of zeolites and intrazeolitic A major technical problem is the overwhelming fluorescence from trace hydrocarbon impurities upon visible excitation.15 Thus, the development of UV excitation for Raman enhancement as described in this paper may alleviate this problem, as has been noted for biological systems.1° Moreover, zeolites are extensively used as catalysts for transformation of aromatic hydrocarbons, and UV resonance Raman spectroscopy has the potential to examine the fate of such molecules in the intrazeolitic environment, with high selectivity and sensitivity.
EXPERIMENTAL SECTION The solution UV absorption spectra and the solid diffuse reflectance spectrum were obtained wing a Shimadzu (Model 265) spectrometer. The liquid samples were excited with 20 mW of 647-nm radiation from a Kr ion laser (Coherent Innova KlOO), and the Raman scattering waa collected, filtered through a Spex 1403 double monochromator, and detected with an RCA C31034 GaAs photomultiplier tube. The normal Raman spectrum of kaminopyridine adsorbed on zeolite NaY was taken with 406.7-nm radiation from the same laser. Resonance Raman spectra were taken using 10 mW of 266.2-nm radiation from 30-H~Quantel
I
245
w
z V
6) Ht4AP/CH30H
2
[t:
$m
< C) 4AP/H20
1
I
250
268
300
NM Flgure 1. Uttravlolet absorptlon spectra of (A) 1 X M 4-amlnopyridine in methenol, (B) 1 X M kminopyridlne with the addklon M 4of concentrated %I (50% by volume), and (C) 0.5 X amlnopyrldlne In water. Figure 1D displays a dlff usareflectance spectrum of 4-amlnopyrMlne adsorbed on zeolite Nay.
YG580 NdYAG laser (the fundamental 1064-nm line was frequency doubled twice using supplied KDP (potassium dihydrogen phosphate) crystals. The radiation was typically spread over a circle of diameter 0.1 cm, leading to peak fluxes of 9 X 105 W/cm2. The scattering was filtered using a Spex 1877 Triplemate monochromator, equipped with a 2400 g/mm grating. The detector was a EG&G PARC Model 1420 Si photodiode array, liquid cooled to -35 "C. Union Carbide (LZY-52)zeolite Y was ion exchanged with 1 M NaCl for 12 h to ensure complete occupation of all cationic sites by Na+ ions. The zeolite was then calcined at 500 "C under a flow of O2 for 12 h to remove any residual organic impurities that would cause background fluorescence. Water vapor was passed over the NaY upon cooling to room temperature to fill all of ita pores with water molecules. Samples were prepared by pressing approximately 150 mg of the above NaY into a pellet at 4000 psi. These pellets were then activated at 500 "C under a vacuum of lo4 Torr for 4 h to remove water and then transferred to a drybox and stored in a sealed vial containing Caminopyridine in methanol for 12 h. Final preparation involved transferring a pellet to a vacuum line, and pulling vacuum ( Torr) for 1 h to remove any solvent methanol,just prior to recording a spectrum.
RESULTS AND DISCUSSION Electronic Spectra of 4-Aminopyridine. Figure 1A displays a UV absorption spectrum of 4-aminopyridine in methanol and consists of a major band at 245 nm (e 14000) with a low-intensity shoulder at approximately 268 nm (6 2400). There have been two assignments for these bands, both involving transitions of the lone electron pairs of the amino group to ?r nonbonding molecular orbitals of benzene.16 And, in one of these assignments, the lone electron pairs are proposed to be delocalized on the benzene ring.16 Upon addition of concentrated HCl, there is a bathochromic shift of the 245-nm band to 263 nm, due to protonation. There is agreement in the literature that the protonation takes place on the ring nitrogen atom.16 This is based on both the red shift in the electronic spectra and the strong basicity of this
- -
ANALYTICAL CHEMISTRY, VOL.
15000
1
64, NO. 8, APRIL 15, 1992
966
Table I. Assignments of the Vibrational Bands of Neutral and Protonated Forms of 4-Amin0pyridine'~,*~.20
s
850
1
1000'
.\
A) 4AP
I
!
I
1 279 1338
ring breathing
850 1000 1059 1217 1338 1613
1059
10000 10000
protonated 4-aminopyridine cm-' assignmenta
4-aminopyridine cm-' assignments
1613
4
ring breathing
deformation deformation ring-N stretching ring stretching C-H C-H
846 lo00 1045 1203 1534 1645
ring breathing ring, C-H, C=N+ ring, C-H, C=N+ ring, C-H, C=N+ C=N+, C=C, NH2 C=N+, C=C, NH2
A) 4AP
20000
n
+J
800
1000
1200 1400 Raman Shift ("1)
1600
1800
I
J
s
1537
Flgure 2. Raman spectra of 4-amlnopyridlnein Its (A) neutral (1 M) and (B) ac#lc forms (0.5 M) (methanol and acMlfied methanol, 647-nm excitation).
-
10000
molecule (pK, 9.17). Spinner and -workers have proposed that upon protonation, a "pyridone" type tautomeric form (11) as compared to the pyridinium ion (I) is formed."
+
I
I
400
I
I
800 1200 Raman Shift (cm-1)
I
I
1600
Flgure 3. Raman spectra of (A) neutral (1 M) and (8) protonated 4-aminopyrMlne (0.5 M), taken at 266.2 nm (methanol and acldlfled
methanol).
-
The electronic band at 263 nm (e 16500) is assigned to the T-T* transition of the conjugated chromophore. When the solvent is switched to H20,the protonated molecule is the major species (Figure 1C). 4-Aminopyridine, with a pK, of 9.17, has sufficient basic strength to be protonated in aqueous solution, but remains essentially neutral in methanol. The diffuse-reflectance spectrum in Figure 1D is that of Caminopyridine adsorbed on the surface of dehydrated zeolite Nay. The spectrum indicates the possible presence of both the neutral and cationic forms, but it is difficult to confirm that since electronic bands are often broadened on solid supports due to inhomogeneous effects.'* Also, the sample under examination is a Na+ exchanged form of zeolite Y, and there should be no strong Bronsted acid sites in the framework. Raman Spectroscopy of 4-Aminopyridine in Solution. The spontaneous Raman spectra of both neutral and protonated species are presented in Figure 2. These spectra have been reported previously and included here for comparis0n.17J9,20The prominent bands of the neutral pyridine at 850,1000, 1059,1217, and 1613cm-' are all a1modes and have been assigned to u12 (trigonal ring breathing), v1 (totally symmetric ring breathing), vl& (in-plane C-H deformation), vga (in-plane C-H deformation), and uh (ring stretching) modes, respe~tively.~~~'~ Among the weaker bands,1338 cm-' has been assigned to ring-N stretch.lg The peaks at 1030 and 1450 cm-' are due to the presence of the solvent methanol. Upon pro-
tonation, the band at 1059 cm-' shifts to 1045 cm-', and the bands at 1613,1217,and 1279 cm-'disappear, while new bands appear at 1203,1263,1378,1534, and 1645 cm-'. Since the molecule assumes a pyridone-like structure (11) upon protonation, the bands can no longer be assigned based on substituted pyridines. Spinner and c o - ~ o r k e r s have ' ~ ~ ~assigned the prominent bands at 846,1000,1045,1534, and 1645 cm-' to in-plane a, modes centered on the ring, with the 1534- and 1645-cm-' bands having C=N+ and C = C stretching motion, mixed with NH2 scissoring. The 1OOO- and 1045-cm-' bands are also composite bands consisting of CH in-plane deformation and C=N+ stretching. The band a t 846 cm-' is assigned to a ring breathing mode."rm These assignments are indicated in Table I. The resonanceRaman spectra of the neutral and protonated forms were obtained with approximately 10 mW of 266.2-nm excitation (Figure 3). This is the first report of resonanceenhanced Raman spectra of these molecules. All of the a1 modes observed in the spontaneous Raman spectrum are being enhanced, though the major intensity occurs in the 15001600-cm-' region. The band at 1275,1358, and 1565 cm-'are assigned to b2modes, the latter two being observed only in the infrared spectrum.1gThese bands are also assigned to ring stretching modes and in-plane C-H deformation. The pattern of resonance enhancement of all the ring-centered modes would argue for the assignment of the electronic band to a ring-centered transition rather than a charge-transfer transition from the exocyclic NH2 group to the ring which should have enhanced the C-N stretching mode at -1338
958
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
i r 1 1
852
22000
1 1
850 1049
1539
18000 1000
1500 Raman Shift (cm-1)
1000
1400
1800
Raman Shift ("1)
Flgure 4. Raman spectrum of 4-amlnopyrMlne adsorbed on zeolite NaY (406.7 nm, 200 mW).
Flgwe 5. Resonance Raman spectrum of the protonated form of 4amlnopyridlne adsorbed on zeollte NaY (266.2 nm, 10 mW).
cm-'. The spectrum of protonated 4-aminopyridine displays
The spectrum is almost identical with the resonance Raman spectrum in Figure 3B, with the exception of the band at 1210 cm-'. This particular vibration also seems to be influenced by adsorption on the zeolite. Thus, even though both forms of the molecule are present in the zeolite, only the vibrational signature of the protonated amine is seen in the spectrum. The acidic groups protonating the 4-aminopyridinemust arise from SiOH groups on the surface or at defect sites on the zeolite. The acidity of SiOH groups on silica is reported to be higher than that of methanoP and is consistent with our recent studies on acidity of dealuminated f a ~ j a s i t e . ~ ~ However these groups are weakly acidic and will not protonate pyridine, the pKa of which is about 4 units lower than that of ita amino derivative. At low levels of weak zeolite acidity, it is expected that several species of a molecule can exist on the surface. This may include both physically adsorbed and chemisorbed species. Vibrational spectroscopy can be used to distinguish between these forms. The advantage of wing resonance Raman is that it is possible to increase sensitivity and selectivity. This preliminary study demonstrates that it is possible to detect low levels of weakly acidic sites on the zeolite and to Selectively examine chemical changes of small molecules adsorbed on zeolites by using UV resonance Raman spectroscopy,
clear resonance enhanced bands at 846,1OOO, 1050,1202,1537,
and 1654 cm-'. The matching of the excitation wavelength with the maximum of the electronic transition led to considerable signal enhancement for the protonated species as compared to the neutral form. Though we did not quantify this effect, in order to obtain spectra with similar S/N, the integration time for the protonated form was 25% of that of the neutral form. All the resonance enhanced bands are characteristic of the C=C, C=N+, and C-C modes, confirming the formation of the pyridone form of the molecule (11) upon protonation. High peak power pulsed sources that are used in this study have been shown to produce spectra due to transienta as well as photoionization and saturation effecta.21s22We took care in obtaining the spectra shown in Figure 3 at the minimum possible power. In the case of the protonated pyridine, where the excitation coincides with the electronic band, all the Raman bands that were observed (Figure 3B) agree with the continuous wave spectrum at 647.1 nm (Figure 2B). This clearly indicates that no new bands due to any transient, photoexcited species are b e i i observed. For the unprotonated pyridine, new Raman bands are observed for similar excitation powers. However, since the excitation is not in direct resonance with the electronic band (248 nm), it is unlikely that in this case any transient photoexcited species are formed. However, based on the data shown here, we cannot exclude saturation effects. Raman Spectroscopy of 4-Aminopyridine on Zeolite Nay. The spontaneous Raman spectrum of Caminopyridine adsorbed on zeolite Nay, taken with excitation at 406.7 nm and displayed in Figure 4, reveals the presence of both protonated and unprotonated species. The bands at 1046,1534, and 1644 cm-' indicate that some of the molecules are protonated on the surface. However, bands indicative of the neutral molecule are also present at 1221and 1608 cm-l. Since the Raman intensity of the protonated band at 1203 cm-l is very small, it is likely that the neutral 1217-cm-I band is shifted to 1221 cm-I through the interaction with the zeolite. By changing the excitation wavelength to 266.2 nm in direct resonance with the protonated form, the spectrum of the protonated molecule can be selectively examined (Figure 5). The presence of bands at 1210,1539,and 1648 cm-' are observed,along with the absence of those at 1221and 1608cm-'.
REFERENCES (1) Bhatla, S. Zeolite Cate!vs/s: Prlnc@k and AppNcetbns; CRC Press, Boca Raton, FL, 1990. (2) Vaughan, D. E. W. Chem. €ng. Prog. 1966, 84, 25. (3) C b n , N. Y.; Degnan, T. F. Chem. €ng. prop. 1990, 84, 32. (4) Benesi, H. A. J . Pnys. Chem. 1957, 61, 970. (5) Beaumont, R.; Barthomeuf, D. J . Catal. 1972. 27. 45. (6) Ward, J. W. J . Catel. 1968, 10, 3. (7) Place. R. D.; Dutta, P. K. Anal. Chem. 1991, 63. 348. ( 8 ) Ward, J. W. I n ZeMe chsmhrby and Cafalys&; Rabo. J. A., Ed; Amerlcen Chemical Society: Washington, D.C.. 1976; p 118. (9) Smlth. J. V. Zeaute Catelmis: Phhclpks and App#cethms, CRC Press: Boca Raton, FL. 1990; p 51. (10) (a) Bloloqicel Appkatbns of Ramen Spectmscopy; Spiro. T. G., Ed.; john Wiley & Sons: New York, 1987; Vols 1-3. (b) Asher. S. A. Annu. Rev. Pnys. Chem. 1966, 39, 537. Mayne, L.; Hudson, 6. J . phys. Chem. 1967, 91, 4438. (c) Kllbugh, P. M.; Devito, V. L.; Asher. S. A. A M . Spectmsc. 1991. 45, 1067. (11) Dutta. P. K.; Zaykodcl. R. I n w ~ Chem. . 1966, 2 4 , 3490. (12) Dutta. P. K.: h i , M. J . RIP. Chem. 1967, 91, 4329. (13) Dutta, P. K.; Zaykoskl, R. J . Mys. Chem. 1869, 93, 2603. (14) Incavo. J. A.; Dutta, P. K. J . phys. Chem. 1990, 94, 3075. (15) Dulta, P. K.; Zaykoskl. R. Zedites 1966, 6 , 179. (16) Mason, S. F. J . Chem. Soc. 1960, 219.
Anal. Chem. 19@2,64, 957-960 (17) Spinner, E. J . Chem. Soc. 1082, 3119. (18) Tape, J.; Tsuruya, T.; Sato, T.; Yoneda, Y. Bull. Chem. Soc. Jpn. 1072, 45, 3609. (19) Shimatla. H.; Ohtoh, T.; Nibu, Y. Fukuoka Unlv. Sci. Rep. 1088, 18 (2). 123. (20) Batts, B. D.; Spinner, E. Aust. J . Chem. 1080, 22, 2595. (21) Bajdor, K.; Nishimura, Y.; Peticoias, W. L. J . Am. Chem. Soc. 1087, 109, 3514.
957
(22) Jones, C. M.; DeVlto, V. L.; Harman, P. A.; Asher, S. A. Appl. Spec-. trosc. 1087, 4 1 , 1268. (23) Hair, M. L.; Hertl, W. J . B y s . Chem. 1070, 7 4 , 91. (24) Dutta, P. K.; Turbeville, W. J . phvs. Chem. 1001, 95, 4087.
RECEIVED
for review September 16, 1991. Accepted J a n W
27, 1992.
Tandem Mass Spectrometric Analysis of Peptides at the Femtomole Level Peter T. M. Kenny and Ron Orlando* Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan
I n the sequentlal analysls of peptldes by fast atom bombardment (FAB) tandem m a r spectrometry (MS/MS), the prlnclpal obstacle to decreaslng sample quantltles was determlned to be the rlgnal-to-background ratio of the ionlzatlon/decrorptlon process. By decresdng the background Ion current, contlnuow-flow FAB allows complete analyds (both MS and MS/MS) to be performed on 25-75% l e r sample than requlred for a conventlonal FABMSIMS experlment alone. The comblnatlon of CF-FAB wlth array detection permltted sequential analysls of several peptider (900-2000 Da) at the 900 hnol to 5.8 pmol level, wlthout Interference from the background. These k v d r do not produce a molecular ton specks easlly dlscernlble above the background In conventlonal FAB.
INTRODUCTION Over the past several years, tandem mass spectrometry (MS/MS)'s2 has played an increasing role in the structural analysis of complex bimole~ules.~ In the case of sequential analysis of peptides, MS/MS offers several advantages over traditional Edman degradations, including the abilities to sequence peptides present in mixtures, identify modified amino acids, and sequence peptides with blocked N termini.3 These advantages often offset the larger sample quantities traditionally required for analysis by MS/MS. Although numerous studies have been performed on reducing the quantity of peptide required for sequencing by MS/MS, probably the most promising new technique is array detection. With the ability to simultaneously detect entire portions of the mass spectrum, array detection has been credited with increasing the sensitivity of MS/MS analysis by a fador of 50-100 over conventional point detection? This development has decreased the sample requirements needed by four-sector MS/MS instruments to the point where they now rival, if not exceed, that of Edman degradations.- With the development of detectors which can simultaneouslyrecord larger portions of the spectrum, further reductions in sample requirements are expected. Although not commonly encountered, artifact peaks have been reported in MS/MS spectra arising from fragmentation of the coselected FAB background ion."" When the sample size is reduced, the intensity of the analyte ion decreases relative to the background leading to increased interference. Eventually, these artifacts dominate the MS/MS spectrum and can obscure all structural information.lOJ1Consequently, the ultimate sensitivity of MS/MS analysis may be imposed 0003-2700/92/0364-0957$03,00/0
by the signal-to-background ratio produced by the ionization process." With the limitation imposed by the isobaric matrix ion, an obvious strategy for reducing sample requirements is to increase the precursor ion intensity relative to the background, which is the focus of this report. By reducing the amount of matrix, continuous-flow FAB (CF-FAB) has been demonstrated to provide a 50-100-fold improvement in the signalto-background ratio obtained in the analysis of peptides at the low-picomole level.12 Similarly, the use of CF-FAB as the ionization process for MS/MS analysis is shown here to yield a 25-75-fold decrease in analyte consumed compared to conventional FABMS/MS. The combination of CF-FAB and array detection decreases the amount of a peptide required for sequence analysis to the femtomole range, a level that is well below that of the stepwise E d " method. Additionally, this strategy allows sequence determination from peptide quantities that do not produce noticeable ions by conventional FAB. EXPERIMENTAL SECTION All mass spectra were acquired with a JEOL (Tokyo, Japan) HX/HXllOA tandem four-sectormass spectrometer, which was operated at 10-kV accelerating potential. Spectra acquired by MS 1are averaged profile data of 12 scans as recorded by a JEOL complement data system. These spectra were acquired from m / z 1000 to 1500 in 6 s at a rate that would scan from m/z 1to 6000 in 1min. A fitering rate of 300 Hz and an approximate resolution of lo00 were used in acquiring these spectra Ions were produced by fast atom bombardment with xenon using a JEOL FAB gun operated at 6 kV, with either a conventional FAB or a FRIT-FAB ion source. In the case of conventional FAB, the samples were diluted in a 1%aqueous solution of trifluoroacetic acid. Aliquota of the sample, 1 pL, were mixed with 1 pL of thioglycerol, the FAB matrix, on the probe tip. For the FRIT-FAB spectra, the samples were dissolved in a 1:l mixture of water and methanol. This solution also contained 10% trifluoroacetic acid and 4 % thioglycerol. The concentrations of peptides were 370 fmol/pL substance P, 580 fmol/pL porcine renin substrate tetradecapeptide, 90 fmol/pL for the synthetic peptide ASHIPRFV-NH,, and 300 fmol/pL for the synthetic peptide SDYEGRLIQNSL. Samples were introduced to the FRIT-FAB source by a 60-pm-i.d. fused-silica capillary tube that was 37 cm in length, which was inserted into the 10-pL micropipet that contained the sample. This combination of capillary size and length was found to introduce the sample at a constant rate of 2 pL/min, without the need for a syringe pump. Approximately 10 pL of the sample solution was sufficient to fill the transfer line, ensure constant flow, tune the mass spectrometer, and acquire both MS and MS/MS spectra. Further decreases in sample volume were not investigated, as this appeared to be the minimum volume needed to wash the sides of the sample tubes, ensuring complete use of 0 1992 American Chemical Society
