1977
Anal. Chem. 1984, 56, 1977-1979
. HO
. OH
C1 during FAB in glycerol (J. A. Kelley, National Cancer Institute, personal communication),indicating the possibility of dehalogenation from nonaromatic carbon.
0
0
HO
OH
HO
ACKNOWLEDGMENT We are grateful to D. L. Smith for conversion of the MAT 731 mass spectrometer for negative ion measurments and to J. Franks for helpful discussions. Registry NO.1,1024-99-3;2,957-75-5;3,2880-89-9;4,316-46-1; 5,146-77-0;6, 2946-39-6;7,4016-63-1;8, 55627-73-1;thyroxine, 51-48-9; glycerol, 56-81-5.
OH
MH
Mx
1, x =I 2, X = Br
3, x =
c1
4,X=F
LITERATURE CITED (1)
cil-5-yl radical is highly reactive and rapidly extracts hydrogen from a structural variety of compounds (16)a t rates which are sufficiently similar to imply little selectivity in the abstraction step. For example, the relative rate constant for H abstraction by the uracil-5-yl radical a t pH 7 for benzene is 16 f 2 and is 12 f 2 for ethanol (17). The photochemical analogy is of added interest because of the finding by White et al. that bombardment of surfaces by neutral or ion beams in the 30 eV-100 keV energy range induces optical emission in the visible, ultraviolet, and infrared regions (18,19). Of perhaps greater significance are radiolysis studies (20) of reactions induced by the solvated electron, generated by y-irradiation, or direct electron bombardment of aqueous solutions. Upon irradiation, 5-bromouracil thus reacts by dissociative electron capture to form the uracil-by1 radical and Br- (15,21, 22)) the former then producing uracil by hyrogen abstraction from the medium. When the reaction with 5-bromouridine is initiated by FAB, both uridine and unreacted nucleoside are observed in the positive ion mass spectrum, while Br- is additionally observed in the negative ion spectrum. The generality of reactions of this type in FAB mass spectrometry has not yet been determined, so two additional polar halogenated compounds were examined for evidence of dehalogenation: thyroxine and 5-(4,6-dichlorotriazin-2-y1)aminofluorescein. The fluorescein derivative (MxH+ = m / z 495) showed no observable dechlorination (MHH+ = m/z 461) measured at resolution 10000 to avoid overlap from m / z 461 of glycerol. On the other hand, thyroxine showed extensive dehalogenation by replacement of both one and two iodine atoms: MxH+, m/z 778,100% relative intensity; M"+, m/z 652,88%; m / z 526, 34%. The overall significance of these studies is twofold. First, the interpretation of FAB mass spectra must take into account the possiblity of processes which are unexpected on the basis of conventional gaseous ion chemistry, as earlier suggested by Field (23).For example, under most circumstances, the finding of ions representing M H from eq 1would lead to the conclusion that the original sample contains uridine. Second, extensive radiation damage to glycerol occrs during FAB (23)) with consequences that are largely unexplored at the present time. Note Added i n Proof. 5'-Chloro-5'-deoxy-5,6-dihydro-5azacytidine was observed to undergo 20% exchange of H for
(1) Rinehart, K. L., Jr. Science(Washington, D.C.) 1982,218,254-260. (2) Burlingame, A. L.; Whitney, J. 0.; Russell, D. H. Anal. Chem. 1984, 56,417R-467R. (3) Harrison, A. G. "Chemical Ionlzatlon Mass Spectrometry"; CRC Press: Boca Raton, FL, 1983. (4) Morris, H. R. "Soft Ionization Biological Mass Spectrometry"; Heyden: Philadelphia, PA, 1981. (5) Pang, H.; Schram, K. H.; Smith, D. L.; Gupta, S. P.; Townsend, L. B.; McCloskey, J. A. J . Org. Chem. 1982,47, 3923-3932. (6) Sethi, S. K.; Smlth, D. L.; McCloskey, J. A. Biochem. Biophys. Res. Commun. 1983, 112, 126-131. (7) Martln, S. A.; Costello, C. E.; Blemann, K. Anal. Chem. 1982, 5 4 , 2362-2368. Crow, F. W.; Tomer, K. 8.; Gross, M. L.; McCloskey, J. A.; Bergstrom, D. E. Anal. Biochem. 1984, 139, 243-262. Reiser, R. W. Org. M s s Spectfom. 1969,2,467-479. McCloskey, J. A. I n "Basic Principles in Nucleic Acid Chemistry"; Ts'o, P. 0. P., Ed.; Academic Press: New York, 1974; Voi. I,pp 209-309. Harrison, A. G.; Lin, P.-H. Can. J . Chem. 1975,53, 1314-1318. Leung, H.-W.; Harrlson, A. G. Can. J . Chem. 1976,5 4 , 3439-3452. Reference 3,pp 102-106. Wang, S. Y. I n "Photochemistry and Photobiology of Nucleic Acids"; Academic Press: New York. 1976:Vol. I.DO 296-31 1. (15) Bhatia, K.; Schuler, R. H. J.'Phys.'Chem.'fS73, 77, 1888-1896. (16) Hutchinson, F. 0.Rev. Biophys. 1973,6 , 201-246. (17) Ebel, R.; Kraljic, 1. Eur. Biophys. Congr., Proc., Ist, 1971, 1971,2, 109-1 14. (18) White, C. W.; Simms, D. L.: Tolk, N. H. Science (Washinaton, D . C . ) 1972, 177, 481-486. (19) White, C. W.; Thomas, E. W.; Van der Weg, W. F.; Tolk, N. H. I n "Inelastic Ion-Surface Collisions"; Tolk, N. H.; Tuliy, J. C.; Heiland, W.; White, C. W., Eds.; Academic Press: New York, 1977; pp 201-252
(20) SchokrG. I n "Effects of Ionizing Radiation on DNA"; Huttermann, J.; Kohnlein, W.; Teoule, R.; Bertinchamps, A. J., Eds.; Springer-Verlag: New York, 1978;pp 153-170. (21) Bansal. K. M.; Patterson, L. K.; Schuier, R. H. J . Phys. Chem. 1972, 7 6 , 2386-2392. (22) Rivera, E.; Schuler, R. H. J . Phys. Chem. 1983,8 7 , 3966-3971 and references therein. (23) Field, F. H. J . Phys. Chem. 1962,8 6 , 5115-5123.
'
Current address: Travenol Laboratories, Inc., 6301 Lincoln Avenue, Morton Grove, I L 60053.
Satinder K. Sethi' Chad C. Nelson James A. McCloskey* Departments of Medicinal Chemistry and Biochemistry University of Utah Salt Lake City, Utah 84112
RECEIVED for review March 16,1984. Accepted May 3,1984. S.K.S. gratefully acknowledges support from National Cancer Institute Training Grant CA 09038. This work was supported by Grant GM 21584 from the National Institute of General Medical Sciences.
Microcolumn Gel Permeation Chromatography with Inductively Coupled Plasma Emission Spectrometric Detection Sir: The increasing need to characterize the components of complex mixtures separated by high-performance liquid
chromatography (HPLC) has encouraged the development of hyphenated techniques such as HPLC-MS (1-9), HPLC-
0003-2700/84/0356-1977$01.50/00 1984 American Chemical Society
1978
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
3
carbon detection. The experimental conditions are as follows: frequency,27.12 MHz; plasma power, 2.2 kW coolant Ar gas flow rate, 16 L/min; plasma Ar gas flow rate, 1L/min; sample Ar gas flow rate, 0.46 L/min; the observation height from the coil, 12 mm; the torch type, Scott's type; nebulizer, cross flow type and its chamber size is 31 mm i.d. X 162 mm; monochromator, 0.5 m KM-1 Ebert mounting type; photomultiplier, Hamamatsu R-456. The micro-HPLC pump was a microfeeder MF-2 (Azuma Electric Co., La., Tokyo, Japan). The column was made of Teflon tubing of 1mm i.d. X 20 cm packed with Fine GEL 92-220 (11.7 km, Jasco, Tokyo, Japan), and mobile phase was distilled water. The test solutes were commercially available products. The cross sectional view of the nebulizer directly connected to the microcolumn is shown in Figure 1. In this figure, the stainless steel capillary tubing (130 pm i.d. X 40 mm) is directly connected to the outlet of the microcolumn by heating Teflon tubing to the melting point and then cooling it to shrink. The eluent from the column is introduced to the nebulizing point (P in the figure). The sample Ar gas carries the eluent as a fog into the plasma torch. It was recognized from some preliminary experiments that the flow rate of the sample Ar gas should be optimized to the range of 0.44-0.46 L/min, because signal intensity of carbon emission line was maximized at this range of Ar gas flow rate. The present system is compared with the previous system (33, 34) in performance. In the previous system, the eluent of micro-HPLC was carried into the ICP nebulizer through the interfacing device with water carrier. The interfacing device was a simple T-type connection of two types of stainless steel tubings: one is the same size as the micro-HPLC column and the other is slightly larger in diameter. This interfacing device was simply connected between the outlet of the microcolumn and the inlet of the nebulizer. The carrier flow rate was 0.50 mL/min and the dilution at the interface was about 25-fold.
I'
Figure 1. Cross sectional view of cross flow nebulizer: (1) eluent inlet from a microcolumn; (2) stainless steel capillary tubing, 130 p m i.d. (310 p m 0.d.) X 40 mm length; (3) glass tubing, 320 p m 1.d. X 30 mm; (4) sample Ar gas Inlet; (5) Teflon nebulizer body; (0) O-ring; (7) nebulized eluent; (P) nebulizing point.
FTIR (10-17), etc. Many of these detection techniques benefit from the low flow rates encountered in microcolumn HPLC (micro-HPLC) (18-20). In terms of atomic spectrometric techniques, inductively coupled plasma atomic emission spectrometry (ICP-AES) is a powerful sensitive and selective detection technique with great potential for combination with HPLC (21-32). Therefore, a coupling of microcolumns to an ICP h w been demonstrated by the authors (33,34), although those studies have a serious drawback in detection sensitivity because the eluent from the microcolumn is diluted with the sample carrier-solvent in the interface device. To improve this low sensitivity of the technique previously described, we have developed a new direct-connecting system between a microcolumn and an ICP nebulizer. All the organic compounds present can be measured with the micro-HPLCICP system by monitoring of carbon emission line at 247.9 nm. For gel permeation chromatography with water as mobile phase, the Ar plasma is maintained stable and the GPC is considered as one of the best examples of the application of the micro-HPLC-ICP detection system. Another reason to develop detection for microcolumn technology is that there are still no refractive-index type detectors like those used for conventional HPLC. The purpose of the present paper is to demonstrate the potential of the newly developed micro-HPLC-ICP system for the analysis of carbon-containing materials by monitoring the carbon emission line.
RESULTS AND DISCUSSION The performance of the total chromatographic system can be evaluated with the help of the so-called van Deemter curve, i.e., the plot of HETP vs. linear mobile phase velocity. Since the velocity is proportional to the flow rate, similar plots can also be prepared by substituting flow rate for velocity. Such a plot is more meaningful in practice because it clearly demonstrates the change in performance (system efficiency) when changing the flow rate. Using raffinose as the model solute in the micro-HPLC-ICP system examined, we have found that the shape of that curve corresponded to those well-known in general HPLC. The minimum HETP was obtained at a flow rate of 4 pL/min, although it is generally considered that the minimum HETP should be obtained at flow rates higher than 4 pL/min for such microcolumn. This observation could be interpreted as being the result that the best combination of eluent flow and sample Ar gas flow is present near at the flow rate of 4 pL/min. Figure 2 shows the results obtained by injecting 1.6 pL of a solution of two three-saccharide mixtures obtained with the previous micro-HPLC-ICP system and the present system. A significant improvement in the resolution and sensitivity is noted with the present system. This improvement is at-
EXPERIMENTAL SECTION A Nippon Jarrell-Ash (Tokyo,Japan) ICAP-500swas used for PREVIOUS SYSTEM
PRESENT SYSTEM
3 2 J
5
1
1
1
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1
-
2
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.
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- J ,1
.
I
1 ,
l
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,
I
/
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I
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I /
0
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I
I
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,
,
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RETENTION TIME (min)
Flgure 2. Carbon selective chromatograms for saccharides with the previous micro-HPLC-ICP system and the present micro-HPLC-ICP system: column, 1 mm i.d. X 20 cm length packed with SC-220 gel; temperature, 80 OC; mobile phase, water; sample carrier in the previous system, water; flow rate of mobile phase, 16 pL/min; sample volume, 1.6 pL; sample, 80 p g of each saccharide as below tor A and C, 160 p g of each saccharide B and D, respectively; (1) raffinose, (2) glucose, (3) arabinose.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
1979
3 2 1
-
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'
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'
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I
d
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I
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RETENTION TIME (min)
20
Figure 4. Carbon selective chromatograms of poly(ethy1ene glycol) (PEG 200) with the micro-HPLC-ICP system: column, mobile phase, flow rate and sample volume, same as in Figure 3; temperature, 40 OC for A, 60 O C for 6, and 80 OC for C, respectively: sample, 10% PEG 200 in water.
-A 20
0
20
I
0
R E T E N T I O N T I M E (mi111
Flgure 3. Carbon selecthre chromatograms for saccharides wlth the micro-HPLC-ICP system: column, temperature and mobile phase, same as In Flgure 2; flow rate of mobile phase, 4 pL/mln; sample volume, 1.6 pL; sample, 12 pg of each saccharide for A, 24 pg for B and 160 pg for C, respectively; (1) raffinose, (2) glucose (3) arabinose.
tained by excluding the dilution at the interfacing device and reducing the extracolumn effect in the connecting tube. In the previous work (33,34),it was very hard to use the system at a flow rate lower than 10 pL/min, but the direct connection system proposed in this work can be used even at 2 pL/min with appreciable sensitivity and good peak shape. With the higher flow rate of mobile phase (above about 15 pL/min) recommended for optimal use of the previous system, it was necessary to compromise chromatographic efficiency for the effect of fast flow rate. However, it is not necessary with the present system. From the above discussion, it is apparent that the micro-HPLC-ICP system proposed here is improved by the direct connection between the microcolumn and the nebulizer. In order to demonstrate the potential of the system, some chromatograms were measured. Figure 3 illustrates chromatograms for the separation of saccharides. Good chromatograms can he detected even at 4 pL/min flow rate, and the signal response depends on the amount of carbon in the sample. Those chromatograms show that few micrograms of saccharides could be easily detected by monitoring carbon with ICP emission spectrometry. The plots of carbon content and the peak heights showed linear relationships in the amount range of 5-200 pg of various saccharides examined. The detection limit of this system was obtained as the amount of raffinose which provided a peak height corresponding to twice the base line noise level, and this value is 800 ng of carbon which is superior to the value obtained by the conventional HPLC-ICP system (a few micrograms carbon) described by Yoshida et al. (32). The second demonstration of this system has been performed with poly(ethy1ene glycol) (PEG 200) as the test solute. In figure 4, the chromatograms of PEG 200 at various column temperatures are shown. Increasing the column temperature increases the resolution. At the column temperature of 80 "C, each oligomer of PEG 200 can be observed as the peak (35). Longer columns could give more highly resolved peaks of each oligomer, although the maximum temperature of these kinds of column materials should be maintained at lower than 90 OC. The micro-HPLC-ICP system works very well for oligomer separations in aqueous gel permeation chromatography. In conclusion, the simple combination of micro-HPLC and ICP-AES for gel permeation chromatography with water as mobile phase was investigated. This technique could be a valid
detection system in microcolumn separations for organic species with non-UV or weak UV absorbance which require any detecting methods other than UV. Direct coupling of capillary columns and ICP is also possible, and the evaluation of this approach is currently in progress. Registry No. 1,512-69-6;2,50-99-7;3,147-81-9; poly(ethy1ene glycol) (SRU), 25322-68-3. LITERATURE CITED (1) Eckers, C.; Games, D. E.; Lewis, E.; Nagaraja Rao, K. R.; Rossiter, M.; Weerasinghe, N. C. A. Adv. Mass Spectrom. 1980, 8B, 1396. (2) Henion, J. D. Adv. Mass Spectrom. 1980, 8B, 1241. (3) Blakely, C. R.; Vestal, M. L. Anal. Chem. 1983, 5 5 , 750. (4) Cairns, T.; Seigmund, E. G.; Doose, G. M. Anal. Chem. 1982, 5 4 , 953. (5) Henion, J. D.; Mayiin, G. A. Blomsd. Mass Spectrom. 1980, 7 , 115. (6) McFadden, W. H.; Bradfoki, D. C. J . Chromatogr. Sci. 1979, 17, 518. (7) Games, D. E.; Lewis, E. Blomed. Mass Spectrom. 1980, 7 , 433. (8) Henion, J. D.: Thomson, B. A.; Dawson, P. H. Anal. Chem. 1982, 5 4 , 451. (9) Hayes, M. J.; Lankmayer, E. P.; Vouros, P.; Karger, B. L.; McGuire, J. M. Anal. Chem. 1983, 55, 1745. 10) Johnson, C. C.; Taylor, L. T. Anal. Chem. 1983, 55, 436. 11) Brown, R. S.; Taylor, L. T. Anal. Chem. 1983, 5 5 , 723. 12) Kuehi, D.; Griffiths, P. R. J . Chromatogr. Scl. 1979, 17, 471. 13) Kuehl, D. T.; Griffiths, P. R. Anal. Chem. 1980, 5 2 , 1394. 14) Jinno, K.; Fujimoto, C.; Hirata, Y. Appl. Spectrosc. 1982, 36, 67. 15) Jinno, K.; Fujimoto, C.; Ishii, D. J . Chromatogr. 1982, 239, 625. 16) Fujimoto, C.; Jlnno, K.; Hirata, Y. J . Chromatogr. 1983, 258, 81. 17) Jinno, K.; Fujimoto, C. Chromatographia 1983, 17, 259. 18) Ishll, D.;Asei, K.: Hibi, K.; Jonokuchl, T.; Nagaya, M. J . Chromatogr. 1977, 144, 157. (19) Novotny, M. Anal. Chem. 1981, 5 3 , 1294A. (20) Novotny, M. Anal. Chem. 1983, 5 5 , 1308A. (21) Fraiey, D. M.; Yates, D.; Manahan, S. E. Anal. Chem. 1979, 5 1 , 2225. (22) Gast, C. H.; Kraak, J. C.; Poppe, H.; Maesson, F. J. M. J . Chromatogr. 1979, 185, 549. (23) Morlta, M.; Uehiro, T.; Fuwa, K. Anal. Chem. 1980, 5 2 , 349. (24) Hausier, D. W.; Taylor, L. T. Anal. Chem. 1981, 5 3 , 1223. (25) Morita, M.; Uehlro, T.; Fuwa, K. Anal. Chem. 1981, 5 3 , 1806. (26) Morita, M.; Uehiro, T. Anal. Chem. 1981, 5 3 , 1997. (27) Fraiey, D. M.; Yates, D. A.; Manahan, S. E.; Stalling, D.; Petty, J. Appl. Spectrosc. 1981, 3 5 , 525. (28) Whaiey, B. S.; Snabie, K. R.; Browner, R. F. Anal. Chem. 1982, 5 4 , 162. (29) Heine, D. R.; Penton, M. B.; Schiabach, T. D. Anal. Chem. 1982, 5 4 , 81. (30) Gardner. W. S.; Landrum, P. F.; Yates, D. A. Anal. Chem. 1982, 5 4 , 1196. (31) Yoshida, K.; Haraguchi, H.; Fuwa, K. Anal. Chem. 1983, 5 5 , 1009. (32) Yoshida, K.; Hasegawa, T.; Haraguchi, H. Anal. Chem. 1983, 5 5 , 2106. (33) Jinno, K.; Tsuchida, H. Anal. Lett. 1982, 15. 427. (34) Jinno, K.; Nakanishi, S.; Tsuchlda, H.; Hirata, Y.; Fujimoto, C. Appl. Spectrosc. 1983, 37, 258. (35) Showa Denko Co., Ltd., Shodex Data Sheet, BJ 8200277 and BJ 8200278, Tokyo, Japan, 1982.
Kiyokatsu Jinno* Shoji Nakanishi Toshiyuki Nagoshi School of Materials Science Toyohashi University of Technology Toyohashi 440,Japan
RECEIVED for review December 27,1983. Accepted April 25, 1984.
