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Anal. Chem. 1990, 62, 1700-1705
in combination with TSP LC /MS is reported here for the fiist time. This work is a sequel to previous research of this group in DLI using a membrane phase separator (13)and of Vouros and co-workers (22) who used a gravity-based separator in combination with a transport interface prior to MS detection. Future work will include the investigation of postcolumn ion-pair extraction prior to TSP MS detection.
ACKNOWLEDGMENT The authors are indebted to the late Professor R. W. Frei for initiating this collaborative research project. R. Alonso from the CID-CSIC is thanked for laboratory assistance. LITERATURE CITED (1) Voyksner, R . D.; Haney, C. A. Anal. Chem. 1985, 5 7 , 991-996. (2) Garteir, D. A,; Vestal, M. L. LC, Liq. Chromatogr. HPLC Mag. 1985, 3,334-346. (3) Barcelb, D. Org. Mass Spectrom. 1989, 2 4 , 219-224. (4) BarcelB, D. Biomed. Environ. Mass Spectrom. 1988, 17, 363-369. (5) Barcelb, 0.; Aibaiggs, J. J. Chromatogr. 1989, 474, 163-173. (6) Covey, T. R.; Bruins, A. P.; Henion. J. D. Org. Mass Specfrom. 1988, 23, 178-186. (7) Smith, R. W.; Parker, C. E.; Johnson, D. M.;Bursey, M. M. J. Chromafogr. 1987, 394, 261-270. (8) Alexander, A. J.; Kebarle, P. Anal. Chem. 1986, 58, 471-478. (9) Barcelb, D. Org. Mass Specifom. 1989, 2 4 , 898-902. (10) Parker, C.E.; Smith, R. W.; Gaskell, S. J.; Bursey, M. M. Anal. Chem. 1986, 58, 1661-1664. (11) Vreeken, R . J.; Brinkman, U. A. Th.; De Jong, G. J.; Barcelb, D. Biomed. Environ . Mass Spectrom., in press.
(12) Barcelb, D. Chromafographia 1988, 25, 295-299. (13) Apffel, J. A.; Brinkman. U. A. Th.; Frel, R. W. J. Chromafogr. 1984, 312, 153-164. (14) Geerdink, R . B.; Maris, F. A.; Frei, R. W.: De Jong, G. J.; Brinkman, U. A. Th. J. Chromatogr. 1987, 394, 51-64. (15) Parker, C. E.; Haney, C. A.; Harvan, D. J.; Hass, J. R. J. Chromatogr. 1982, 242, 77-96. (16) Barcelb, D.; Maris, F. A.; Geerdink, R. B.; De Jong, G. J.; Brinkman, U. A. Th. J . ChrOmatOgr. 1987, 394, 65-76. (17) De Ruiter, C.; Wolf, J. H.; Brinkman. U. A. Th.: Frei, R. W. Anal. Chim. Acta 1987, 192, 267-275. (18) De Ruiter, C. Thesis, Pre- and post column fluorescence derivatization in HPLC, Free University, Amsterdam, The Netherlands, 1989; pp 75-88. (19) Schill. G.;Modln, R.;Borg, K. 0.; Persson, B. A. I n Handbook of Derivatives for Chromatography; Blau, K., King, G. s., Eds.; Heyden: London, UK, 1977; Chapter 14. (20) Voyksner, R . D.; Bursey, J. T., Pelllzari, E. D. J. Cbromafogr.1984, 312,221-235. (21) Stan, H. J.; Kellner, G. Biomed. Mass Specfrom. 1982, 9 , 463-492. (22) Vouros, P.; Lankmayr, E. P. Hayes, M. J.; Karger, B. L.: McGuire. J. M. J. Chromatogr. 1982, 251, 175-188.
RECEIVED for review December 11,1989. Accepted March 23, 1990. Financial support was provided by NATO Research Grant 0059f 88, the Ministerio de Educacion y Ciencia, the Netherlands Foundation for Chemical Research (SON) with financial acid from the Netherlands Organization for the Advancement of Scientific Research (NWO) (Grant No. 700.344.006),and the Commission of the European Communities fellowship (ST2*-0488).
Effects of Peptide Hydrophobicity and Charge State on Molecular Ion Yields in Plasma Desorption Mass Spectrometry Rong Wang, Ling Chen, and Robert J. Cotter* Middle Atlantic Mass Spectrometry Facility, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Plasma desorption mass spectra were obtained for a series of peptides, grouped in four mass ranges having approximately 9, 20, 30, and 40 amino acid residues. Within each group, the individual peptides differed in hydrophobicity, charge state, and retention time, as measured on a revenred-phasg HPLC column. Comparison of the molecular ion intensities in the positive ion mass spectra of peptides from each group showed a strong dependence upon hydrophobicity and no correlation with charge state. Plasma desorption mass spectra of mixtures of ail the peptides within each mass range generally resulted in the desorption of a single residue and suppression of the ion slgnai from other components. I n most cases, this could be correlated with hydrophobicity, as calculated from the Bull and Breese index; however, a better correlation existed when the results were compared with reversed-phase retention times. I n general the spectra of mixtures were not Influenced by charge state (except in the absence of hydrophobic peptides), as the same component in each peptide mixture produced the most abundant ions in both positive and negative ion spectra.
INTRODUCTION An effective strategy for the analysis of peptides and proteins with known, or expected, amino acid sequences is the
direct mass spectrometric analysis of their unfractionated enzymatic digests using fast atom bombardment ( I , 2) or plasma desorption mass spectrometry (3). These survey spectra provide a series of peaks whose masses can be correlated directly with those calculated from the sequence for peptide fragments resulting from enzymatic cleavage at specific locations on the protein. The masses observed in such spectra can be and have been used to locate posttranslational and chemical modifications, to reveal oxidation of methionine to the sulfoxide ( 4 ) ,to verify the formation and/or location of disulfide bonds (5,6),and to determine sequence variations (7). FAB mass spectral analysis of complex mixtures often results in preferential desorption of the more surface-active (8) or hydrophobic (9) components. Naylor et al. (9) have shown that the selective desorption of peptide fragments in a tryptic digest can be correlated with their average hydrophobicities calculated from the Bull and Breese indexes (10) and noted that the presence of hydrophobic fragments in a tryptic digest effectively suppresses the desorption of the more hydrophilic residues. While the selectivity observed in the FAI3 technique is related to diffusion of sample to the surface of the liquid matrix, Nielsen and Roepstorff (11) have proposed that the PDMS analysis of tryptic digests (desorbed from the solid state on a nitrocellulose-coated foil) is relatively insensitive to hydrophobicity and found no correlation between the selective desorption of peptides in an enzymatic digest with their
0003-2700/90/0362-1700$02.50/00 1990 American Chemical Society
rlALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
Bull and Breese indexes. Instead, they found that selectivity was related to the charge state of the individual peptides at pH 7. They therefore suggested that peptides containing several basic residues will dominate the positive ion mass spectrum, while more negatively charged peptides will be observed in the negative ion spectrum. Recently, researchers at Genentech (4) reported the tryptic mapping of the 23-kDa (Da = dalton) recombinant DNAderived human growth hormone (rhGH) by fast atom bombardment, for which 21 peptides had been observed by reversed-phase HPLC. These same peptides were all observed in the FAB mass spectrum, with the exception of one of the very hydrophilic peptides and a high molecular weight (3762 Da) disulfide-linked fragment. This same sample was then examined in our laboratory by plasma desorption mass spectrometry (12) to compare the differences in selectivity. In this case, the unfractionated digest was adsorbed to a nitrocellulose foil, and plasma desorption mass spectra were obtained before and after washing with deionized water. It was noted that washing improved the ion signal for the larger and more hydrophobic peptides, including the disulfide-linked fragment a t 3762 Da. In addition, the most abundant peaks in the positive ion spectrum were also most abundant in the negative ion spectrum, suggesting relative insensitivity to the charge state. While these results suggested that washing may result in the removal of small and hydrophilic peptides, we were motivated by the insensitivity to charge state to reexamine the factors that effect the magnitude of the ion signal. Because the size (MW) of the peptides appeared to be a factor in our previous experiment with the digest of recombinant human growth hormone, we attempted a more controlled study using peptides of similar mass. Also, because the Bull and Breese scale was used in previous studies by Roepstorff et al. ( I I ) , we used that scale as well; however, we note that calculations of relative hydrophobicities from the Bull and Breese scales (10) may be inaccurate for smaller peptides, since the hydrophilic contribution of the terminal amino and carboxy groups becomes more significant than for larger peptides. Conversely, the secondary and tertiary structures of larger peptides determine which residues are exposed on the peptide surface and may result in hydrophobicities that cannot be correlated with a calculation that assumes a linear structure. Thus, we examined a series of peptides grouped in four mass ranges. In addition, since the retention times of peptides purified by reversed-phase HPLC also reflect their hydrophobicities, these were also determined and compared with the ion signals from each of the peptides analysed singly and in mixtures with other peptides of similar mass.
EXPERIMENTAL SECTION Peptides used in this study were purchased from Sigma (Kalamazoo, MI), trifluoroacetic acid (TFA) from Pierce Chemical Co. (Rockford, IL), acetone (reagent grade), acetonitrile (HPLC grade), and HPLC water from J. T. Baker Chemical Co. (Phillipsburg, NJ), and nitrocellulose membrane (0.45 pm) from Bio-Rad (Richmond, CA). Ultrapure water was made in our laboratory by distillation, ion exchange, and redistillation. Peptide samples were prepared as 1 nmol/pL solutions in aqueous 0.1% TFA. In each case, 5-pL samples were applied to the sample foil, which was then'covered with a microscope cover glass for 3 min to reduce solvent evaporation as the peptides reach equilibrium between the solvent and nitrocellulose surface. The cover glass was then removed, carrying away most of the solvent, while the solvent remaining on the foil was allowed to evaporate. In some cases, plasma desorption mass spectra were obtained directly from these samples. In other experiments, the sample foil was then rinsed with 1mL of aqueous 0.1% TFA to remove Na+ and K+ contaminants and weakly desorbed peptides, prior to analysis by PDMS. The sample sizes and concentrations used were those reported by Nielsen et al. (13) to be optimal for the
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Table I. Amino Acid Sequences of Peptides Used in This Study A1 thyrotropin releasing hormone: WAGGDASGE A2 [Arg8]-vasopressin:CYFQNCPRG-NH2 A3 [T~r~l-bradykinin: RPPGFSPYR A4 dynorphin,-,: YGGFLRRIR A5 Des-Asp1-angiotensinI: RVYIHPFHL B1 katacalcin: DMSSDLERDHRPHVSMPQNAN B2 @melanocytestimulating hormone (human): AEKKDEGPYRMEHFRWGSPPKD B3 parathyroid hormonezH8 (human): LQDVHNFVALGAPLAPRDAGS B4 bovine adrenal medulla docosapeptide: YGGFMRRVGRPEWWMDYQKRYG C1 parathyroid hormone,M8 (human): APLAPRDAGSQRPRKKEDNVLVESHEKSLG C2 C-pe~tide~-~~: EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ C3 corticotropin releasing factor antagonist: DLTFHLLREMLEMAKAEQEAEQAALNRLLLEEA-NH2 C4 growth hormone releasing factor,+ (amide):
HADAIFTSSYRRILGQLYARKLLHEIMNR-NH2 D1 growth hormone releasing factorl4 (human): YADAIFT-
NSYRKVLGQLSARKLLQDIMSRQQGESNQERGA D2 adrenocorticotropic hormone (human): SYSMEHRFWGK-
PVGKKRRPVKVYPNGAEDESAEAFPLEF
D3 urotensin (teleost fish): NDDPPISYDLTFHLLRNMIEM-
ARIENEREQAGLNRKYLDEV-NHo
D4 Tyr-corticotroph releasing factor (human and rat):
Y SEEPPISLDLTFHLREVLEMARAENLAQQAHSNRKLMEII-NH, ~
nitrocellulose technique. That is, molecular ion yields were maximized in this concentration range and were relatively insensitive to small changes in concentration. Mass spectra were obtained on a BIO-ION, Nordic AB (Uppsala, Sweden) BIN 10 K plasma desorption mass spectrometer, equipped with a 10-pCi 252Cfsource and a PDP 11/73 data system. The accelerating voltage was +18 kV for the positive ion mass spectra and -16 kV for the negative ion spectra. Retention times for each of the peptides were determined on a Beckman (Berkeley, CA) Model 114 binary gradient system with a Rheodyne (Cotati, CA) 7125 injection valve fitted with a 2 0 4 sample loop, a Supelco (Bellefonte, PA) LC-304 analytical reversed-phase column (250 x 4.6 mm, 5 pm), and an Applied Biosystems (Ramsey, NJ) Model 757 variable UV detector fitted with a high-pressure flow cell. The mobile phases used in this study were (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile. A linear gradient was programmed from 100% A ( t = 0) to 50% A/5070B (t = 60 min) at a flow rate of 0.5 mL/min. Retention times were measured at ambient temperature.
RESULTS A series of 17 peptides were examined in this study, grouped according to the number (9, 20, 30, and 40) of amino acid residues. Their sequences are shown in Table I; their molecular weights, Bull and Breese hydrophobicities, net charges, and retention times are shown in Table 11. Instinctively, one would expect peptides containing basic residues to form positive ions. The question then becomes whether the presence of several basic residues or the ratio of basic to nonbasic residues should result in more abundant positive (or negative) ion yields for one peptide in relation to another. One measure of the basicity of a peptide is its isoelectric point, or its net charge at pH 7 as used by Nielsen and Roepstorff (11). At the same time, solution pH has been shown to be a critical factor in both FAB and PDMS mass spectrometry. Schronk and Cotter (14) have noted that the pH of solutions of bovine insulin, prior to mixing with glycerol, effect the ratios of singly, doubly, and triply charged molecular ions observed in their fast atom bombardment mass spectra. More appropriately, Silly and Cotter (15) have studied the effects of the pH of the solution deposited on nitrocellulose foils on the plasma desorption mass spectra of peptides with
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
1702
MIXTURE of 61, 62, 83 and 84
Table 11. Physical Properties of Peptides Used in This Study
(POSITIVE ION)
6000
peptide
MW
hydrophobicity”
A1 A2 A3 A4 A5 BI B2 B3 B4 C1 C2 C3 C4 D1 D2 D3 D4
848.9 1084.3 1076.3 1137.4 1181.4 2436.7 2661.0 2148.5 2839.4 3285.8 3020.4 3826.6 3473.2 4544.3 4541.3 4869.7 4920.9
+443 +lo7 -94 -262 -544 +270 +160 +30 -86 +217 +lo1 -77 -160 +lo2 +8 -79 -140
net charge retention p H 7 p H 1.9 time -2 +2 +2 +3 +1 -2 0 -1 +3 +I -5 -4 +4 +2 +1 -3 -2
+1 +2 +3 +4 +4 +5 +7 +3 +6 +8 +1 +5 +8 +7 +9 +7 +7
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Figure 2. Positive ion plasma desorption mass spectrum of a mixture of peptides containing 20 amino acids.
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400
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Flgure 1. Molecular ion signalhoise ratios of peptides as a function of their hydrophobicities (expressed as cal/mol) calculated from the Bull and Breese indexes ( 70). (To achieve similar scales, SIN data for the 9, 20,30, and 40 amino acid peptides were divided by 4, 1.7, 2, and 1, respectively.)
different isoelectric points. They found that the mass spectral peak widths were narrowest when the solution pH was close to the isoelectric point. They also found that protonated molecular ion yields were maximized when the solution deposited on the nitrocellulose foil was prepared l or 2 pH units below the value of the isoelectric point. Thus, while such samples are indeed dried prior to mass analysis, it has been clear that preparation of samples in acidic solutions is critical to the formation of protonated molecular ions. With that in mind, net charges in Table I1 have been calculated a t pH 7 , as used by Nielsen and Roepstorff ( I I ) , and a t pH 1.9, the approximate pH of the 0.19i TFA solutions from which the samples were deposited. Mass Spectra of Single Peptide Solutions. The molecular ion yields (measured as the ratio of the molecular ion signal to the background signal) from the mass spectra of each of the peptides as a function of their Bull and Breese hydrophobicities are shown in Figure 1. Within each molecular weight group, the molecular ion yields are generally greater for the more hydrophobic (negative index) peptides. After rinsing with aqueous 0.1% TFA (data not shown), the dependence of molecular ion signal on hydrophobicity within each mass group is qualitatively similar, with the more hydrophobic peptides resulting in larger ion signals. The major effect observed is the decrease in ion signal of the low-mass
200
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1do0
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3600
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Figure 3. Negative ion plasma desorption mass spectrum of a mixture of peptides containing 20 amino acids.
(nine amino acid) peptides, consistent with our previous observations for the tryptic mapping of recombinant human growth hormone (12). Because this was true for all subsequent experiments, the results reported below are all those that were obtained without additional washing. At the same time, no correlation was observed between relative ion signal and the charge state at either pH 7 or 1.9. Mass Spectra of Peptide Mixtures with Similar Mass. The positive ion plasma desorption mass spectrum of an equimolar mixture of the 20 amino acid peptides (Bl-B4) is shown in Figure 2. Both singly and doubly charged iond of the most hydrophobic peptide (B4, calculated from the Bull and Breese indexes) are observed, while all other components are suppressed. The negative ion mass spectrum (Figure 3) of the same mixture produced a single peak corresponding to the same hydrophobic peptide. A second mixture of 20 amino acid peptides, which did not contain the most hydrophobic peptide, was prepared, and its positive ion mass
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990 MIXTURE of 81, 82 and
MIXTURE of C1, C2, C3 and
(POSITIVE ION)
83
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1703
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I
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100
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ld00
2000
2500
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Figure 4. Positive ion plasma desorption mass spectrum of a mixture
MI2
Figure 6. Positive ion plasma desorption mass spectrum of a mixture of peptides containing 30 amino acids.
of peptides containing 20 amino acids, without the most hydrophobic peptide. MIXTURE of D1. D3 and 04
MIXTURE of C1, C2,C3 and C4
(NEGATIVE ION)
(POSITIVE ION)
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Figure 7. Negative ion pbsma desorption mass spectrum of a mixture of peptides containing 30 amino acids.
Figure 5. Positiie ion plasma desorption mass spectrum of a mixture of aeotides containina 40 amino acids.
spectrum obtained (Figure 4). These peptides are all hydrophilic (positive index) and are all observed, although the peptide with intermediate hydrophilicity (B2) gave the most intense signal. The ion signal in the positive ion mass spectrum was, however, about an order of magnitude lower than that for the B4 peptide in the previous mixture, and no peaks were observed in the negative ion spectrum. In this mixture of hydrophilic peptides, it is possible that the more positive charge on B2 (at both pH 7 and 1.9) contributes to its larger ion signal. The positive ion plasma desorption mass spectrum of a mixture of 40 amino acid peptides (Dl, D3, and D4) is shown in Figure 5. In this case some precipitation in the 0.1% TFA solution was observed and is most likely D4, which is very hydrophobic and, therefore, less soluble in aqueous solution. An ion signal is, however, observed for D4, but a much larger
signal is observed for D1, the most hydrophilic peptide. At pH 1.9 all three peptides have the same charge state; at pH 7 D1 is the only positively charged peptide. Figures 6 and 7 show, respectively, the positive and negative ion plasma desorption mass spectra for a mixture of 30 amino acid peptides (Cl-C4). The predominant peak observed in both mass spectra is that due to desorption of C3, which is not the most hydrophobic peptide based upon the Bull and Breese scale. We note, however, that the peptide C3 has an exceptionally long retention time and may (in fact) be more hydrophobic than C4. The peptide C4 is observed in the positive ion mass spectrum but not in the negative ion spectrum. While this may be attributed to the fact that it is positively charged at both pH 7 and 1.9, we note that the predominant species (C3) in both spectra is negatively charged at pH 7 and also that the ion signal in the negative ion mass spectrum is sufficiently reduced that the peak due to C4 cannot be observed.
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
o /
so
20
10
M
M
R m W n O N TlYE OF PEPnDE (mln)
Figure 8. Molecular ion signal/noise ratios of peptides as a function of their retention times determined by reversed-phase HPLC. (To achieve similar scales, SIN data for the 9 and 20 amino acid peptides were divided by 4 and 1.2, respectively.)
DISCUSSION From these data we believe that it is possible to make several general observations concerning the influence of both charge state and hydrophobicity on molecular ion signal and the suppression of ion signal in mixtures of peptides. First, when peptides of high hydrophobicity are present in a mixture, the most hydrophobic peptide will give the most intense ion signals in both positive and negative ion mass spectra, irrespective of charge state. In this case, the ion signal from other peptide components may be completely suppressed. Second, retention times obtained on a reversed-phase HPLC column may provide a more accurate measurement of actual hydrophobicity than can be calculated on the basis of the Bull and Breese or other scales. This appears to be the case for the 30 amino acid peptides, in which the major peak results from the species with the highest retention time, and a smaller peak is observed for the peptide with the next highest retention time. Thus, the correlation of molecular ion yield with reversed-phase liquid chromatographic retention time is illus-
trated in Figure 8. In general, retention times reflect mass as well as hydrophobicity, i.e., the larger peptides tend to have longer retention times. For the 20, 30, and 40 amino acid peptide groups, the molecular ion yields are nearly linear with retention time, while there is somewhat less agreement for the smaller peptides. We have noted earlier that calculated hydrophobicities may be less accurate for smaller peptides, and it may be that similar difficulties may be encountered in estimating relative hydrophobicities from retention times. Conversely, it may be that smaller peptides will exhibit a poorer correlation with hydrophobicity and that other factors contribute to the strength of the ion signal. In contrast to previous studies ( I I ) , however, which suggested that hydrophobicity did not play a significant role in the relative ion yields in plasma desorption mass spectrometry, the general trends shown in Figure 8 would seem to suggest otherwise. In evaluating the two methods for estimating relative hydrophobicities, it is interesting to compare directly the hydrophobicities calculated from the Bull and Breese indexes with the measured retention times. The plots shown in Figure 9 would suggest that the calculated hydrophobicities are most accurate for the peptides containing about 20 amino acids. For the larger peptides, hydrophobicities calculated from the Bull and Breese scales are poorly correlated with retention time, reflecting (perhaps) the influence of secondary and tertiary structure in determining which residues are exposed to the solvent. Third, in mixtures in which the peptides are largely hydrophilic, selectivity is then influenced by the charge state.
CONCLUSIONS This is a somewhat more complex picture than that presented by Nielsen and Roepstorff (11) and provides a much greater (even overriding) influence by hydrophobicity. At the same time, dependence upon hydrophobicity is not surprising, since the desorption of peptides is (in general) enhanced by the use of nitrocellulose, a surface with a high affinity for hydrophobic residues. FAB and PDMS mass spectrometry of enzymatic digests can be viewed as techniques that are complementary to tryptic mapping by HPLC. In the latter technique, peptides that coelute reflect their similar hydrophobicities and cannot be
65
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50
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CALCULATED HYDROPHOBICITY
Figwe 9. Retention times of peptides determined by reversed-phaseHPLC as a function of their hydrophobicities (expressed as cal/mol) determined by the Bull and Breese indexes ( 70).
Anal. Chem. 1990, 62,1705-1709
distinguished. Conversely, a PDMS or FAB mass spectrum of a even a crude fraction containing peptides of nearly equal hydrophobicity would be likely to produce ion signals from all of the components, which would be distinguished by their masses. For example, Tsorbopoulos et al. (16) were able to record most of the tryptic peptides from recombinant human growth hormone by PDMS analysis of HPLC fractions containing peptides with similar retention times. Thus, for PDMS as well as FAB, an understanding of the important role of hydrophobicity is essential to the development of effective protocols for determining peptide and protein structure.
LITERATURE CITED (1) Biemann, K. In Methods in Protein Sequence Analysis; Elzinga, M., Ed.; Humana Press: Clifton, NJ, 1982. (2) Morris, H. R.; Panico, M.; Taylor, G.W. Biochem. Biophys. Res. Commun. 1983, 717, 299. (3) Craig, A. G.;Engstrom, A.; Bennich, H.; Kamensky, I . Biomed. Environ. Mass Spectrom. 1987, 74, 669. (4) Canova-Davis, E.; Chloupek, R. V.; Baldonado, I. P.; Battersby, J E.; Spellman, M. w.; Baca, L. J.; O'COnnOr, B.; Pearlman, R.; Ouan, C.; Chakel, J. A.; Stults, J T.; Hancock, W. S . Am. Biotech. Lab. 1988, May, 8-77. (5) Raschdorf, F.: Dahinden, R.; Maerki. W.: Richter, W. J.; Merryweather,
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J. P. Biomed. Environ. Mass Spectrom. 1988, 76, 3. Burman, S.; Wellner, D.; Chalt, B.; Chaudhary, T.; Breslow, E. R o c . Natl. Acad. Sci. U . S . A . 1989. 86, 429. Naylor, S.; Ang, S.-0.; Williams, D. H.; Moore, C. H.; Walsh, K., Biomed. Environ. Mass Spectrom. 1989, 18, 424. Barber, M.: Bordoli, R. S.; Elliot, G. S.; Sedgwick, R. D.; Tyler, A. N. J . Chem. SOC.,Faraday Trans. 1983, L79, 1249. Naylor, S.; Findeis, A. F.; Gibson, B. W.; Willkms, D. H. J . Am. Chem. SOC. 1986, 708, 6359. Bull, H. B.; Breese. K. Arch. Biochem. Biophys. 1974, 767, 665. Nielsen, P. F.: Roeostorff. P. Blamed. Envlron. Mass Smctrom. 1989. 78, 131. Chen, L.; Cotter, R . J.; Stults, J. T. Anal. Biochem. 1989, 783, 190. Nielsen, P. F.; Klarskov, K.; Hojrup, P.; Roepstorff, P. Blomed. Endron. M s s Spectrom. 1988, 17, 355. Schronk, L.; Cotter, R. J. Biomed. Mass Spectrom. 1986, 13, 395-400. Silly, L.; Cotter, R. J. J . Phys. (France) 1989, 12.7, 37-40. Tsarbopoulos, A.; Becker, G. W.; Occolowitz, J. L.; Jardine, I . Anal. Biochem. 1988, 171, 113.
RECEIVED for review November 1, 1989. Accepted April 23, 1990. This work was supported by grants from the National Science Foundation (DBM 85-15390) and from the National Institutes of Health (RR 02727). Mass spectral analyses were carried out at the Middle Atlantic Mass Spectrometry Laboratory, an NSF Shared Instrumentation Facility.
TECHNICAL NOTES Automated Preconcentration of Trace Metals from Seawater and Freshwater Alexander van Geen*J and Edward Boyle Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 INTRODUCTION King and Fritz (1)recently pointed out that separation and concentration of trace metals from sample matrices by solvent extraction (2) is a labor-intensive procedure that is not easily automated. In response, these workers developed an alternative method based on the adsorption of complexed metals onto a resin column. Sodium bis(2-hydroxyethyl) dithiocarbamate (NaHEDC) was shown to form water-soluble hydrophobic complexes with several metals (Cd, Co, Cu, Ni, Pb, and Zn). These complexes adsorbed quantitatively on XAD-4 resin when seawater or distilled water, spiked at concentrations on the order of lo4 M, was passed through a column. A reliable method capable of processing a large number of samples was required for our work on the distribution of trace metals in coastal waters (3). For this reason we automated a version of the King and Fritz method, which was modified in key aspects to allow for analyses at typical natural dissolved metal concentrations M). With this procedure, ten 30-mL samples can be preconcentrated with only 1h of operator attention; the complete procedure is completed in less than 4 h. The nonautomated manipulations include preparations for a set of extractions and sample loading. The sample throughout rate (samples preconcentrated per hour) is not higher than could be achieved by an experienced worker using solvent extraction, but the analyst's productivity is much higher with the present method since other work can be done while preconcentration is underway. The device is constructed for the most part from commercially available components and could be adapted easily to other column separation Current address: Department of Civil Engineering, Stanford University, Stanford CA 94305.
techniques requiring trace-metal-clean conditions.
EXPERIMENTAL SECTION Apparatus. The main components of the preconcentration device are illustrated in Figure 1. A system of 10 columns and 10 sample reservoirs, including both air and reagent manifolds with their respective control valves, fits in a laminar flow-bench 2 ft X 2 ft X 2 ft in size (EnvironmentalAir Control, Inc.). Unless laboratory dust levels are excessive, it is not necessary to work in a completely enclosed clean room in order to obtain acceptably low contamination levels using this preconcentration technique, particularly since most of the apparatus is sealed. Each of the ten extraction units is built around a custom-designed three-way L-shaped valve block fitted with the Teflon cylinder from a Nalgene stopcock (no. 6470). The work reported here was done with custom blocks made of polypropylene; later experience indicates that fabrication is easier when these blocks are made from ultrahigh molecular weight polyethylene. Maximum operating pressure is 20 psi. Valve blocks include four reagent outlets compatible with standard chromatography fittings (1/4-28) used for l/g in. 0.d. Teflon tubing. The following connections are made to each valve (Figure 2a): (1)A polypropylene-syringe sample reservoir (Becton Dickinson & Co.; choice of volume, 10-60 mL) using a Tefzel Luer-lock + 1/4-28 adapter. Each syringe-barrel-reservoir is capped by a section of another syringe mounted upside down. A in. indentation is milled into the bottom barrel reservoir; the truncated upper section is fitted into this groove, and epoxy glue is applied on the outside of the joint to maintain an air seal against interior pressure. To avoid contamination, the analyte should not reach the level of this connection. Each reservoir cap is attached to a common air manifold via a small three-way stopcock and a Luer connection. (2) A small resin column (0.3 mL) made of a section of '/g in. 0.d. (0.06 in. i.d.) Teflon tubing. The resin is supported by a double
0003-2700/90/0362-1705$02.50/00 1990 American Chemical Society
