1236
Anal. Chem. 1984, 56,1236-1240
of the unknown species. The general applicability of the moving belt interface and the nebulization approach was further demonstrated by analyzing the same sample in a different laboratory by using a normal bore chromatographic separation. It thus should be possible to obtain consistent data by on-line HPLC/MS from one laboratory to another. Registry No. 1,5-DIOH-NAP, 83-56-7;resorcinol, 108-46-3; phenol, 108-95-2; guaiacol, 90-05-1; 3-methylphenol, 108-39-4; 4-methylphenol, 106-44-5; 2-methylphenol, 95-48-7; dimethylphenol, 1300-71-6; 4-ethylphenol, 123-07-9; catechol, 120-80-9; water, 7732-18-5.
LITERATURE CITED (1) Arplno, P. J.; Guiochon, G. J. Chromatogr. 1982, 251, 153. (2) Alcock. N. J.; Eckers, C.; Games, D. E.: Games, M. P. L.; Lant, M. S.; McDowali, M. A.; Rossiter, M.; Smith, R. W.; Westwood, S. A.; Wong, H. Y. J. Chromatwr. 1982. 251. 165. Blakely, C. R.; Vesbl, M. L.'Ana/. Chem. 1983, 55. 750. Smith, R. D.; Burger, J. E.; Johnson, A. Anal. Chem. 1981, 53,1603. Hardln, E. D.; Fann, P. P.; Blakely, C. R.; Vestal, M. L. Anal. Chem. 1984, 58,2. Smith, R. D.; Johnson, A. Anal. Chem. 1981, 53,739. Hayes, M. J.: Lankmayr, E. P.; Vouros, P.; Karger, B. L.; McGuire, J. M. Anal. Chem. 1983, 55, 1745. Games, D. E.; Lant, M. S.; Westwood, S.A.; Cocksedge, M. J.; Evans, N.; Williamson, J.; Woodhall, B. J. Biomed. Mass Spectrom. 1982, 9 ,
215. Forster, M. G.; Meresz, 0.; Games, D. E.; Lant, M. A. Biomed. Mass Spectrom. 1983, IO, 338.
S.;Westwood, S.
(IO) Takeuchi, T.; Hirata, Y.; Okumura, Y. Anal. Chem. 1978, 50, 659. (11) Henion, J. D.; Wachs, T. Anal. Chem. 1981, 53, 1963. (12) Schwartz, H. E.; Karger, B. L.;Kucera, P. Anal. Chem. 1983, 55, 1752. (13) Harvey, M. S.;Stearns, S.D. "Liquid Chromatography In Envlronmental Analysls"; Lawrence, J. F., Ed.; Humana Press: Clifton, NJ, 1982; Chapter 10.
(14) Vouros, P.; Biemann, K. Org. Mass Spectrom. 1970, 3 , 1317. (15) Games. D. E.; Hewling, M. J.: Westwood, S. A.; Morgan, D. S. J. Chromatogr. 1982, 250,62. (16) Lauer, H. H.; Rozlng, G. P. Chfomatographia 1981, 14, 641. (17) Scott, R. P. W.; Simpson, C. F. J. Chromatogr. 1982, 20,62. (18) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55,730. (19) Taylor, G. R o c . R . SOC. London, Ser. A 1953, 279, 186. (20) Reese, C. E.; Scott, R. P. W. J. Chromatogr. Sci. 1980, 18, 479 (21) Sparacino, C. M.; Minick, D. J. Envlron. Sci. Techno/. 1980, 14, 880. (22) Alcock, N. J.; Eckers, C.; Games, M. P. L.; Lant, M. S.;McDowali, M. A.; Rossiter, M.; Smith, R. W.; Westwood, S. A,: Wong, H. T. J. J. Chromatogr. 1982, 251, 165.
RECEIVED for review November 29, 1983. Accepted March 19, 1984. This work was supported by funds from the U.S. Environmental Protection Agency, Cooperative Agreement CR807325-02. The contents do not necessarily reflect the views and policies of the Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. This is Contribution No. 186 from the Barnett Institute of Chemical Analysis.
Determination of Nonderivatized Peptides by Thermospray Liquid Chromatography/Mass Spectrometry David Pilosof, Hee Yong Kim, Douglas F. Dyckes, and Marvin L. Vestal*
Department of Chemistry, University of Houston, Houston, Texas 77004
The potentlal of thermospray mass spectrometry for on-line detection of peptides separated by liquid chromatography Is demonstrated. By use of thermospray lonlzatlon, peptides produce mass spectra with abundant protonated (M -t H)' and alkali-attached Ions without fragmentation. With thls method lt Is posslble to observe multiply charged Ions as well. I n the case of peptldes the net charge of the species Is detennlned by the presence of lonlzabie amlno ackl side chains. This effect permits the observation of molecules with molecular weights substantlally above the mass scan ilmit of our quadrupole mass spectrometer, about 1300 amu. By proper adjustment of the solutlon pH we were able to observe the triply and quadruply charged molecule of nonderivatlzed glucagon (mol wt 3483).
The use of combined liquid chromatography/mass spectrometry (LC/MS) has continuously increased in the last decade and has emerged as a powerful analytical tool for samples that are either too thermally labile or too involatile to be amenable to gas chromatography/mass spectrometry (GC/MS) (1-4). Although many interfaces for the direct introduction of samples eluting from a liquid chromatograph (LC) into the ion source of a mass spectrometer have been proposed (3,4),the detection of peptides, in particular those with mol wt >1000, is still a difficult challenge for most of the exisiting LC/MS instruments (3).
One approach for increasing the volatility of peptides has been to block terminal amino and carboxylic groups, and in some cases also the ionic and polar side chains, by chemical derivatization (46).This procedure has been successful for low molecular weight peptides but still has not produced large peptides volatile enough to be subject to electron impact (EI) or chemical ionization (CI) mass spectrometry. Characterization of nonderivatized peptides by mass spectrometry has been performed efficiently by field desorption (FD) by 252Cfplasma desorption (252Cf-PD)(8), and in particular by fast atom bombardment (FAB) (9, 10). The first two methods require off-line sample preparation, incompatible with direct coupling to LC, and the latter has recently been proposed as an on-line detector of LC eluants using the moving belt system (11). The combination with chromatographic separation adds an important dimension to the analysis of impure samples and samples in biological matrices and, in particular, for the detection of oligopeptide mixtures resulting from enzymatic digestion used in peptide mapping. Developments in the methodology of LC separation of peptides and proteins have been reviewed extensively
(a,
(12-14).
Thermospray, defined as the complete or partial vaporization of a liquid stream by heating as it flows through a capillary, has recently been demonstrated as a versatile interface for combined LC/MS (15-18). The heat is supplied electrically and controlled by a feedback system to maintain a constant level of vaporization. The optimal temperature
0003-2700/84/0356-1236$01.50/00 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
is a function of the solvent composition, the flow rate, the capillary dimensions, and the sample to be analyzed (18). The most important analytical advantages of thermospray LC/MS are the ability of the interface to handle conventional LC/MS flow rates, even for aqueous solvents, and the ability to detect nonvolatile and thermally labile compounds such as drugs (16), amino acids (19,20),glucuronides (21),nucleosides, and nucleotides (22). Other advantages are the ease of operation and the simple adaptability of the interface to existing GC/MS instruments. In this work we evaluate the performance of a thermospray interface for the mass spectrometric detection of peptides in solution. Special interest is focused on the capability of the method to detect peptides bearing multiple net charge, which are preformed in solution depending on the pH.
EXPERIMENTAL SECTION Apparatus. The thermospray LC/MS instrument used a slightly modified Biospect quadrupole mass spectrometer with a Finnigan Incos Data System described earlier (16). The most recent thermospray interface consists of a commercial stainless steel capillary (0.015 mm i.d.) to which a high current is applied between two points separated by approximately 30 cm. The electrically heated capillary reaches a temperature sufficient for an almost complete vaporization of the flowing liquid stream. A thermocouple provides feedback to the power supply, which is regulated to maintain the appropriate heat input. This control is particularly useful for temperature regulation against flow fluctuation introduced by the LC pump, or when a gradient between two solvents of very different boiling points is used. All the solutions were delivered by a Constametric 111-Gradient Master Milton Roy HPLC pump. Solutions were injected with a 7010 Rheodyne injector. The peptide separation was done with a reversed-phase ODS 3 pm (4.1 mm X 5 cm) column manufactured by Custom LC, Inc., Houston, TX. Reagents. Angiotensin 11, [Sar1,Ala8]angiotensin11, a-melanocyte stimulating hormone (a-MSH), and glucagon were obtained from Sigma Chemical Co. (St. Louis, MO) and were used without any further purification. Renin substrate was purchased from Boehringer Mannheim (Indianapolis, IN) and also was used without any additional purification. Two synthetic peptides, Thr-Thr-Orn-Gln-Orn-Tyr-NH2 and Thr-Thr-Arg-Gln-ArgTyr-NH2,were prepared in our laboratory and their synthesis will be published elsewhere (23). All the peptides were dissolved in deionized water at lo4 M and were stored frozen. Trypsin, obtained from Sigma Chemical Co. (St. Louis, MO), was purified by ion-exchange chromatography in order to remove the chymotryptic contamination (24). Only the P-trypsin form was used. Trypsin was immobilized at pH 8.0 on glycophase activated controlled pore glass beads, with 125-177 Mm bead size and 500 A nominal pore diameter, from Pierce Chemical Co. (Rockford, IL). Trypsin immobilization was performed following the manufacturer's specifications (25). The total content of trypsin in the column was 5.6 X lod8mol. The solvent used to transport the samples into the thermospray ion source was 0.1 M ammonium acetate, pH 5.5. In cases involving peptide separation by HPLC, this solvent was used with a 30-50% acetonitrile gradient. RESULTS AND DISCUSSION Studies on a-MSH Tryptic Fragments. In the course of our studies to determine the amino acid sequence of peptides (19, 20), we have explored the enzymatic cleavage of peptides into smaller fragments which can be subsequently detected by thermospray LC/MS. Our model compound for these initial studies was a-MSH, whose amino acid sequence is given in Figure 1. The tryptic digestion of a-MSH (mol wt 1665) produces two fragments, an octapeptide (mol wt 1098) and a pentapeptide (mol wt 585) (Figure 1). Such fragmentation is expected since trypsin cleaves peptides at the carboxylic terminus side of arginine and lysine, except in the case where proline is the contiguous amino acid residue. Consequently, for a-MSH, the bond -Lys-Pro- cannot be
1237
U-MSHIMW=1665)
9
-
CH,C-SER
-
TYR- SER MET-GW
- HIS - PHE - ARG ? TAP-GLY-LYS-PROTI
VAL-NH,
TZ
REL.INT. 100.0
49RB
33 IM+ZS)"
a
50.0
600
400
loo0
8W
1203
M/Z
REL INT 743424
1000
I R + HIt
4 d . l
U-YSH
4FTER 5min INCU84TION IN TRYPSIN COLUMN
b IT1 +tH
400
600
800
loo0
1200
M/Z
Figure 1. Mass spectrum of a 4 nmol a-MSH sample. Figure la was obtained without incubating the peptide in a trypsin column. The resuit for the tryptic hydrolysis after a 5-min incubation in the immobilized enzyme column is shown in Figure Ib. The resolution of the quadrupole mass analyzer was insufficient to distinguish masses after the decimal point.
cleaved and hydrolysis of only the -Arg-Trp- bond is expected. Initially 4 nmol samples of a-MSH were injected into an immobilized trypsin column at pH 8.0. After digestion for a determined amount of time with the flow in the column stopped, the column contents were flushed by a flowing buffer into the thermospray ion source. Figure l a shows the spectrum of the a-MSH sample that was not injected through the trypsin column, and therefore was not subjected to enzymatic hydrolysis. The peaks shown correspond to (M + 2H)2+,(M + Na + H)2+,and (M K + H)2+with calculated m / z at 833.5, 844.5, and 852.5, respectively. The spectrum also shows evidence of the presence of (M + 2Na)2+at 863.5. The sodium and potassium adducts originate from trace concentration of the alkali metal ions in the buffer solution. Since the singly charged molecular peaks for (M + H)', (M + Na)+,and (M + K)+ at m/z values of 1666, 1688, and 1704 were all above the mass scan limit of our quadrupole instrument, about 1300, their presence could not be directly monitored. The peak at m / z 556 corresponds to the triply protonated molecular ion. The spectrum shown in Figure l b resulted after a 5-min incubation of a 4 nmol a-MSH sample into the trypsin column.
+
1238 MI2
372 368
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
8 REL. INT.
THR-1WR-ORN-GLN-ORN-TYR-NHp 1M.W. 736)
M/Z
REL INT
r
50'2
83 I 836
h
THR-THR-ARG-
409 413
GLN-ARG-TYR-NHI
879
fM.W. s . 8 2 2 )
881
/
922
[SAR',AU']
929
0
ANGIOTENSIN II 1M.W 9261
b3
a1 I
I
I
I
40
80
I20
160
c -
200
0
TIYEISC)
Flgure 2. Mass chromatograms of an arginine containing hexapeptide and the ornithine peptide from which it was produced. One-nanomole samples were sequentially injected and the corresponding signals were obtained within 10 s. Both peptides were monitored as the doubly protonated ions. A sample of the arginine-containing peptide and the corresponding ornithine analogue were injected at times (a) and (b), respectively. Full mass scans were taken and the regions of interest were selected by computer acqulsltion.
This was determined independently to correspond to approximately 60% digestion. In addition to the residual aMSH at m / z 833, the spectrum shows peaks corresponding to both fragments resulting from the tryptic cleavage. The pentapeptide shows a peak for the protonated molecule at m/z 586, and also alkali metal ion adducts at m / z 608 and 624, respectively. The octapeptide can be detected both as a singly charged ion at m l z 1099 and as a doubly charged ion at mlz 550, 561, and 569, respectively. Since both fragments are present at the same concentration, the larger signal for the pentapeptide is probably a result of its relatively higher volatility. As seen in Figure lb, the presence of alkali adducts can help to differentiate singly charged from multiply charged species. For a singly charged species a sodium and a potassium adduct show up 22 and 38 amu higher than the protonated molecule, whereas doubly charged species show these peaks at 11 and 19 amu higher, respectively. By use of a highresolution instrument, it should also be possible to distinguish multiply charged species since the isotope peaks show up l / n amu apart, where n is the charge of the ion. For the a-MSH doubly charged ion for example, the minimum resolution needed to determine the charge directly would be 1667. Such resolution cannot be obtained with our present quadrupole mass spectrometer. Molecular Weight Determination of Synthetic Peptides. An extremely useful application of thermospray mass spectrometry is the determination of molecular weights of synthetic peptides. In order to corroborate the composition of a synthetic peptide, amino acid analysis is normally applied. When accurate results are needed, this procedure usually requires overnight incubation of the peptide in a strong acid solution at high temperature for the complete hydrolysis of all peptide bonds. After some additional sample preparation, the amino acid analyzer requires a 30-90 min cycle to determine the amino acid composition. Mass spectrometry is advantageous due to the short time needed for the analysis. This is particularly true with the thermospray LC/MS system described in this work. Since no separation column is needed in this case, the sample delivery virtually operates as a flow injection analysis system, with peak widths around a few seconds. Since consecutive samples can be injected every 30 s, a high sample throughput can normally be obtained. This analytical method is of
c
h
100
200
1000
300
TIMEISECI
Separation of peptides by reversed-phase HPLC. A 30-50% acetonitrile gradient in 0.1 M ammonium acetate, pH 5.5, was used; flow rate was 1.0 mL/min. The mass chromatograms correspond to the mass region of interest selected by the computer from full mass scans. Figure 3.
particular value in monitoring the purification of a synthetic peptide. The components separated from a synthetic mixture can be analyzed rapidly in order to determine which ones correspond in molecular weight to the desired product. This is especially advantageous when these components are separated on-line by HPLC. In addition to product identification, an assessment of product purity is possible. The example shown in Figure 2 consists on the verification of a total conversion of Thr-Thr-Orn-Gln-Orn-Tyr-NHz into Thr-Thr-Arg-Gln-Arg-Tyr-NHz. This was obtained by reacting the former hexapeptide with 0-methylisourea in order to convert the ornithine residues into arginine (23). Such conversion results in the increment of the molecular weight by 42 amu per ORN, by adding CN2H, to the side chain. The peptide molecular weight then increases from 738 to 822 amu. Figure 2 shows then mass chromatograms for separate injections of these peptides as the doubly charged ions. Both the mass chromatograms and the corresponding spectra indicated a total conversion of ornithine to arginine. Detection of Peptides Separated by HPLC. A mixture containing 2 nmol each of three peptides, was separated with a reversed-phase column, with a 30-50% acetonitrile gradient in 0.1 M ammonium acetate buffer, Figure 3. The computer-generated mass chromatograms, correspond to the doubly charged molecular ions for a-MSH and renin substrate, at m/z 833 and 881, respectively, and the protonated molecule for [Sarl,Alas]angiotensin I1 at 927 amu. The mass spectrometer was scanned continuously from 400 to 1000 amu in 3 s. Since the peaks obtained in the chromatogram have peak widths around fhwm = 15-20 s, then each signal is defined by five to seven data points. The base line noise arises from the low resolution and the high multiplier voltage (4.2 kV) needed to detect peptides with our quadrupole mass spectrometer. However the spectra obtained after the background is substracted show clear peaks devoid of a significant background, even when injecting subnanomolar amounts. Detection of Glucagon. Glucagon (mol wt 3483) is a single chain peptide composed of 29 amino acid residues. The reason for investigating the performance of the thermospray mass spectrometer for the determination of glucagon was to determine the ability of the method to generate gas phase ions of high molecular weight by direct extraction from solution. With 0.1 M ammonium acetate pH 5.5 as the carrier buffer, no response was obtained by scanning the mass spectrometer up to its mass limit of about m/z 1300. The triply protonated
ANALYTICAL CHEMISTRY, VOL. 56,NO. 8, JULY 1984 REL INT
22688
IM+SH))I 1162
IW 0 2 nmolar GLUCAGON
iM W 34831 IN TFAlpH I851
50 0
600
700
8W
900
loo0
1100
1200
M/Z
Flgure 4. Mass spectrum of a 2 nmoi injection of glucagon in tri-
fluoroacetic acid at pH 1.85. molecular ion has a m / z value of 1162. Its absence at pH 5.5 is explained from the primary structure of glucagon, which contains three aspartic acid residues, negatively charged at this pH, in addition to the positively charged lysine, histidine, and arginine residues. An overall charge of 3+ or 4+ is therefore highly unlikely. The absence of any peak indicates that these highly charged ions are not favorably formed by gas-phase proton exchange either. When 0.1 M trifluoroacetic acid (TFA) pH 1.85 was used as solvent, it was possible to observe both the triply and the quadruply charged ions at m/z 1162 and 872, respectively. At this pH it is also possible to protonate the terminal carboxylic group of tyrosine, pK, = 2.2. With all the four carboxylic groups protonated, the three basic residues and the terminal amino group can produce a quadruply charged ion. The spectrum obtained for a 2 nmol injection is shown in Figure 4. Ions bearing lesser net charge cannot be observed directly due to the instrumental mass scan limitation. The broad peaks observed in the spectrum are the result of the presence of sodium and potassium adducts and the low resolution needed in order to increase the transfer efficiency of the quadrupole. From these results it becomes evident that the thermospray mechanism is capable of producing gas-phase ions of up to 3500 amu regardless of the net charge. Likewise, a peptide with a high relative number of acidic amino acid residues should show multiply charged anionic species, observable in the negative mode. A titration experiment was tried in order to verify the dependence of the relative sizes of the peaks obtained from multiply charged ions on the solution pH. For example, the lower the pH value, the more favored a high positive net charge would be due to protonation. The preliminary results obtained by monitoring the singly and doubly protonated ions of [Sar1,Ala8]angiotensin I1 and a synthetic nanopeptide, Leu-Asn-Val-Val-Thr-Arg-His-Arg-Qr-NH2 (23),surprisingly showed that the relative peak magnitudes for the two specified charge states did not vary over the pH range 1.7-8.0. This experiment seems to indicate the existence of a more elaborate gas phase proton transfer mechanism which is not directly dependent on the solution pH and therefore cannot be predicted simply by considering the solution pK, values of the peptide amino acid residues. More experiments will be developed in order to provide better understanding of the nature of this mechanism. Doubly charged peptides (26) and diquaternary ammonium salts (27) have been reported with field desorption. The detection of multiply charged ions by thermospray mass spectrometry correlates with the theoretical model that suggests that the electrostatic fields produced in a small droplet
1239
electrolytic solution can be strong enough to eject ions preformed in solution. Indeed, these fields were calculated to be comparable in magnitude to the fields obtained in field desorption (28).
CONCLUSIONS The use of thermospray mass spectrometry, described here as a chromatographic peptide detector, is advantageous over more conventional detectors since it provides molecular weight information and, unlike most mass spectrometric methods, it is entirely amenable to on-line coupling and does not impose severe flow rate and solvent composition limitations on the separation process. In this paper we have also demonstrated the ability of thermospray ionization to generate gas phase ions of nonderivatized large polar molecules which are otherwise particularly hard to detect by a mass spectrometer directly coupled to a liquid chromatographer. Although simple peptide mixtures can be analyzed directly, as the example of the tryptic digestion shows, on-line coupling to liquid chromatography is certainly of much wider applicability. This analytical procedure can be extremely useful in monitoring peptide synthesis, in peptide mapping for amino acid sequence determination, and in the detection of peptides in biological matrices. ACKNOWLEDGMENT We thank Michael 0. Perlman for kindly providing the synthetic peptides whose analysis is reported here. Registry No. a-MSH, 37213-49-3; Thr-Thr-Om-Gln-OmTyr-NH2,89556-46-7;Thr-Thr-Arg-Gln-Arg-Tyr-NH2, 89556-47-8; renin substrate, 11002-13-4;[Sarl,Alas]angiotensin11,11130-03-3; glucagon, 9007-92-5. LITERATURE CITED Arpino, P. J.; Gulochon, G. Anal. Chem. 1979, 57, 682A. Majors, R. E. LC,Liq. Chromatogr. HPLC Mag. 1983, 7 (8), 488. Games D. E. Biomed. Mass Spectrom. 1981, 8 , 454. Nibbering, N. M. M. J . Chromatogr. 1982,257, 93. (5) Morris, H.R. F€BS Lett. 1972,22, 257. (6) Yu, T.J.; Schwartz, H.; Giese, R. W.; Karger, B. L.; Vouros, P. J . Chromtogr. 1981,278, 519. (7) Matsuo, T.; Matsuda, H.; Katakuse, I.; Wada, Y.; Hayashi, A,; Fujita, T. Biomed Mass Spectrom 1981 8 25. (8) Hakansson, P.; Kamensky, I.; Sundqvist, B.; Fohlam, J.; Peterson, P.; McNeal, C. J.; MacFarlane, R. D. J . Am. Chem. SOC. 1982, 704, 2948. (9) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N.; Garner, G. V.; Gordon, D. B.; Tetier, L. W.; Hider, R. C. Biomed. Mass Spectrom. 1982,0, 265. (10) Rinehart, K. L. Science 1982,278, 254. (11) Lewis, I . A. R.; Brooks, P. W. Presented at the 186th National Meeting of the American Chemical Society, Washlngton, DC, Aug 29, 1983; paper 26, Anaiytlcal Chemistry Section. (12) Regnler, F. E. Anal. Chem. lg83, 55, 1298A. (13) Alvarez, V. L.; Roitsch, C. A.; Henrlksen, 0. Immunol. Methods 1981, 2 , 83. (14) Seiber, J. N.; Nelson, C. J.; Benson, J. M. J . Chromatogr Sci. 1981, 76 (Steroid Anal. HPLC), 45. (15) Blakley, C. R.; Carmody, J. J.; Vestal, M. L. Anal. Chem. 1980, 5 2 , 1636. (16) Blakley, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750. (17) Vestal, M. L. Int. J . Mass Spectrom. Ion Phys. 1983, 4 6 , 193. (18) Vestal, M. L. Presented at the 186th Natlonal Meeting of the American Chemical Soclety, WashlnQton, DC, Aua 29. 1983. .DaDer . 5, Analvticai Chemistry Section. (19) Kim, H. Y.; Pilosof, D.; Vestal, M. L.; Dyckes, D. F.; Kitcheli, J. P.; Dvorin, E. Proceedings of the 8th American Peptide Symposium, Tucson, AZ, May 1983.(20) Pilosof, 0.; Kim, H. Y.; Vestal, M. L.; Dyckes, D. F. Biomed. Mass Spectrom ., in press. (21) Liberato, D. J.; Fenselau, C. C.; Vestal, M. L.; Yergey, A. L. Anal. Chem. 1983,55, 1741. (22) McCioskey, J. A.; Edmonds, C. G.; Presented at the 186th National Meeting of the American Chemical Society, Washington, DC. Aug 29, 1983, paper 6, Analytical Chemlstry Section. (23) Perlman, M.; Dyckes, D. F.. manuscript in preparation. (24) Walsh, K. A. I n “Methods in Enzymology”; Perlman, G. E., Lorend, L., Eds.; Academic Press: New York, 1970; Vol. XIX, pp 41-64. (25) Plerce Technical Bulletln. (26) Katakuse, I.;Matsuo, T.; Wollnik, H.; Matsuda, H. Org. Mass spectrom. 1979, 74, 457. (27) Heller, D. N.; Yergey, J.; Cotter, R. J. Anal. Chem. 1983, 55, 1310. (1) (2) (3) (4)
.
.
I
I
-
1240
Anal. Chem. 1984, 56, 1240-1242
(28) Vestal, M. L. Proceedings of the 2nd International Workshop on Ion Formation from Organic Solids, Muster: Benninghoven,A., Ed.: Sprlnger: Berlin, 1983;Springer Series in Chemical Physics, pp 246-263.
RECEIVED for review January 12, 1984. Accepted February
27, 1984. We wish to express our gratitude to the National Institutes Of for their support (Grant GM 28291). Parts of this paper were presented at the 186th ACS National Meeting, Washington, DC, Aug 1983, papers 5 and 8, Analytical Chemistry Section.
Adsorption Isotherms of Polycyclic Aromatic Hydrocarbons on Asbestos Chrysotile by High-pressure Liquid Chromatography Hugues MBnard,* Luc Noel, Frank M. Kimmerle, Lyne Tousignant, and Maryse Lambert
Department of Chemistry, Universite' de Sherbrooke, Sherbrooke, Qu&bec,Canada J l K 2R1
A new technique to evaluate the adsorptlon of polycyclic aromatic hydrocarbons (PAHs) from solution In toluene on asbestos flber was studied by liquid chromatography. The length of the new column used Is only 3.5 cm and the applied flow Is 1 mL mln-'. The pressure applled on this column at 25 O C is about 18 MPa (2600 psi). The range of PAHs In the samples injected Into the column was from 10 nmol to 1000 nmoi, except for phenanthrene whlch was up to 10 pmol. We estimated Henry's constant at 25 and 40 OC and we noted that benzo[a]pyrene is more adsorbed, comparatively,than the other PAHs. We demonstrated the influence of the HPLC solvent dryness. We estimate an error of less than 5 % in reproduciblllty with another column. By this method It Is possible to produce an adsorption Isotherm more easily and rapidly than by static adsorptlon. The method may be used to test the carclnogenic potential of chemlcaliy treated fibers.
Polycyclic aromatic hydrocarbons (PAHs) constitute an important class of chemical pollutants, many of which are known or suspected carcinogens and mutagens ( 1 , 2 ) . Several industrial sources of these pollutants have been identified and another important source of these pollutants in the air is known to be tobacco smoke (3). PAHs are among the main identified carcinogenic agents in polluted air and in tobacco smoke. Benzo[a]pyrene (B[a]P) has historically been used as an indicator of the environmental presence of PAHs but it should be stated that it may be subject to atmospheric transformations which severly limit the utility of B[a]P data in risk assessment ( 4 ) . It is very important for us to ascertain if PAHs can be adsorbed on the surface of dust particulates such as on asbestos. We will not describe the mechanism by which dust particles probably adsorbed PAHs, but we only observe the behavior of asbestos with different PAHs. The term asbestos is applied to a group of natural mineral fibrous silicates such as chrysotile, crocidolite, amosite, etc. (5). These fibers are resistant to corrosion, heat, acids, weathering, etc., and their uses are very widespread. Unfortunately, it is now known that inhalation of asbestos fibers can cause very severe medical problems including asbestosis and mesothelioma (6, 7). Synergetic effects of some organical micropollutants when they are associated with asbestos pollution (8) have been clearly demonstrated. In this paper we will expose PAHs to chrysotile fibers under HPLC conditions. 0003-2700/84/0356-1240$01.50/0
EXPERIMENTAL SECTION Apparatus. The equipment used for this study included a Beckman HPLC, Model 100-A, coupled with a Altex Hitachi UV detector, Model 100-40,with a flow cell volume of 20 wL,a strip chart recorder (Varian Model 9176), and a integrator (HewlettPackard Model 3390 A). We have also used a 10.01 "C thermostated bath (Haake Model FE-2) and a refractive index (RI) detector Altex, Model 156, with an 8-pL cell. To fill the column we used a Haskel air-driven fluid pump. The column tubing, 4.6 mm i.d., is cut to a length of 3.5 cm with an internal diameter of 4.6 mm. Reagents. Benzo[a]pyrene (>98%), anthracene (199.9%), pyrene (299%),chrysene (95%), and fluoranthene (298%) were purchashed from Aldrich Chemical Co., Inc. Purified naphthalene was purchased from Fischer Scientific Co., phenanthrene (295%) from BDH Co., and fluorene from Eastman Kodak Co. (Caution: All of these products are potentially carcinogens (I).) Asbestos chrysotile of 369 pm average length and 12.9 m2 g-' of specific surface was isolated from Paperbestos 3 as described previously (9). Toluene and n-hexane were Accusolv-grade from Anachemia Co. Toluene was dried by molecular sieves (Type 3-A,Grade 564) from Fisher Scientific Co. Procedure. All solvents used for HPLC were treated by bubbling dried nitrogen gas in a 4-L bottle for a period of 15 min and then adding 40 g of molecular sieves. The solvents were vacuum degassed for 15 min. Asbestos chrysotile was heated at 110 "C for 6 h and put in a desiccator. The column and the precolumn were first dry packed with chrysotile fibers and then high pressure (52 MPa) was applied by an Haskel pump filled with n-hexane. The column was then put inside a thermostated casing and connected to the HPLC instrument. Eluent flow rate was 1 cm3 min-l. Different PAH concentrations from stock 5 X lo-' M solutions were prepared, except for phenanthrene which was prepared from a 5 x IO-' M stock solution. A liquid chromatograph was used with a sample loop of 20 pL, and the corresponding amounts injected were 10 nmol to lo00 nmol for the PAHs and 10000 nmol for phenanthrene. Chromatographic Measurements. Chuduk's method (IO) was used for the determination of adsorption isotherms. By this method, the curve of the adsorption isotherm is deduced according to the form of the extended side of the chromatographic peak under equilibrium conditions. Gibbs' adsorption value, ri,can be calculated by eq 1(i indicates the product) 1 ri = A
Cl
o
VR dci
where VR is the column retention volume (cm3per column), A is the surface area of adsorbent in the column (m2),and ci is the concentration of adsorbate in the mobile phase on the considered 0 1984 American Chemical Society
