Anal. Chem. 1989, 61, 491-493
stable and easier to tune than those obtained with chirp excitation. Generally, the same pulse width and height can be used for all ions, and isotope ratios are more stable and precise than with chirp excitation experiments performed under the same conditions. This may result because with impulse excitation all ions in the cell are accelerated simultaneously in a very short period of time (less than a microsecond). Another feature of impulse excitation is that the electronic circuitry and computer software needed for impulse excitation are far simpler than are needed for chirp excitation or SWIFT. Chirp excitation requires a computer-controlled frequency synthesizer that can scan a frequency range of several megahertz in a time period of just a few milliseconds. The chirp excitation signal must be highly stable and reproducible from scan to scan so that repetitive scans can be summed together coherently to improve the signal-to-noise ratio of the measurement. In contrast, an impulse excitation amplifier needs only a trigger pulse from the computer and simple adjustments for the amplitude and duration of the pulse. Impulse excitation is fundamentally different from the rf burst and rf chirp excitation methods used previously in FTMS because it is a nonselective, nonoscillatory excitation means. As a result, one of the limitations of impulse excitation is that double resonance ejection and sweep-out experiments, which are so useful for studying ion-molecule reactions, cannot be done because it lacks the necessary mass selectivity. However, there is no reason why these selective, resonance experiments cannot be done in the conventional manner while impulse excitation is used for the FTMS detection of the ions.
ACKNOWLEDGMENT We thank Dr. Carlito Lebrilla for assistance in using the external ion source FTMS instrument a t the University of California, Irvine. LITERATURE CITED Gross, M. L.; Rempel, D. L. Sclence (Washlngton, L X ) 1984, 226, 261-268. Freiser, 8. S. Talenta 1985,3 2 , 697-708. Laude, D. A,, Jr.; Johlman, C. L.; Brown, R. S.; Weil. D. A.; Wilkins, C. L. Mass Spectrom. Rev. 1986,5 , 107-166. Russell, D. H. Mass Spect”. Rev. 1986,5 , 167-189. Ijames. C. F.; Wilkins, C. L. J. Am. Chem. SOC. 1988. 110, 2687-2688. - - -. - - - -. Hunt, D. F.; Shabanowitz, J.; McIver, R. T., Jr.; Hunter, R. L.; Syka, J. E. P. Anal. Chem. 1985,57, 765-768. Hunt, D. F.; Shabanowk, J.; Yates, J. R.. 111; McIver, R. T., Jr.; Hunter, R. L.; Syka, J. E. P.; Amy, J. Anal. Chem. 1985, 57, 2733-2735.
49 1
(8) McCrery, D. A.; Ledford. E. D., Jr.; Gross, M. L. Anal. Chem. 1982, 108, 1435-1437. (9) Sherman, M. G.; Kingstey, J. R.; Hemminger, J. C.; McIver, R. T., Jr. Anal. Chlm. Acta 1985, 178, 79089. (10) Sherman, M. G.; Land, D. P.; Hemminger. J. C.; McIver, R. T., Jr. Chem. Phys. Lett. 1987, 137, 298-300. (11) Land, D. P.; Tai. T . I . ; Llndquist. J. M.; Hemminger, J. C.; McIver, R. T., Jr. Anal. Chem. 1987. 5 9 , 2924-2927. (12) Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 26, 489-490. (13) Marshall. A. G.; Roe, D. C. J. Chem. Phys. 1980, 7 3 , 1581-1590. (14) Huang, S. K.; Rempel, D. L.; Gross, M. L. Int. J. Mass Spectrom. Ion Processes 1986, 72, 15-31. (15) Kopel. P.; Allemann, M.; Kellerhals. H.P.; Wancrek, K. P. Int. J. Mass Spectrom. Ion Processes 1986, 74, 1-12. (16) van der Hart, W. J.; van de Guchte. W. J. Int. J. Mass Spect”. Ion Processes 1988p8 2 , 17-31. (17) Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 2 5 , 282-283. (18) Ledford, E. B., Jr.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984,5 6 , 2744-2748. (19) Rempel, D. L.; Ledford, E. B., Jr.; Gross, M. L. Anal. Chem. 1987,5 9 , 2527-2532. (20) Marshall, A. 0.; Wana. T.C. L.; Rlcca. T. L. J. Am. Chem. SOC. 1985,107, 7893-7asi7. (21) Wang, T.C. L.; Ricca, R. L.; Marshall, A. 0. Anal. Chem. 1986,5 8 , 2935-2938 -- - - - - - -. (22) Chen, L.; Wang, T.-C. L.; Rlcca, T. L.; Marshall, A. G. Anal. Chem. 1987. 5 9 , 449-454. (23) Sharp, T. E.; Eyler, J. R.; Li, E. I n t . J. Mass Spectrom. Ion Phys. 1972. 9 , 421. (24) Hunter, R. L.; Sherman, M. G.; McIver, R. T., Jr. Int. J. Mass Spectrom. Ion Phys. 1983,50, 259-274. (25) Comisarow, M. B. J. Chem. Phys. 1978, 6 9 , 4097-4104. (26) McIver, R. T., Jr.; Hunter, R. L.; Ledford. E. B., Jr.; Locke, M. J.; Francl, T. J. Int. J. Mass Spectrom. Ion Phys. 1981, 3 9 , 65-84. (27) McIver, R. T.. Jr.; Hunter, R. L.; Story, M. S.; Syka. J.; Labunsky. M. Paper presented at the 31st Annual Conference on Mass Spectrometry and Allied Topics, Boston, MA, May 6-13, 1983. (28) McIver, R. T., Jr.; Apparatus and Method for Injectlon of Ions into an Ion Cyclotron Resonance Cell. US. Patent 4,535.235. Aug 13, 1985. (29) McIver. R. T., Jr.; Hunter, R. L.; Bowers, W. D. I n t . J. Mass Spectrom. Ion Processes 1985. 6 4 , 67-77. (30) McIver, R. T., Jr.; Baykut, G., Hunter, R. L., unpubilshed results.
Robert T. McIver, Jr.* Department of Chemistry University of California Irvine, California 92717
Richard L. Hunter Gokhan Baykut IonSpec Corporation 17951 Skypark Circle, Suite K Irvine, California 92714 RECEIVED for review August 22, 1988. Accepted November 11, 1988.
Separation of Dansylated Methylamine and Dansylated Methyl-&-amine by Micellar Electrokinetic Capillary Chromatography with Methanol-Modified Mobile Phase Sir: Recent papers have expanded the versatility of capillary electrophoresis by utilizing micellar solutions in micellar electrokinetic capillary chromatography (MECC) (I,2). The technique has been applied to such separations as phenylthiohydantoin amino acids (3),B6Vitamers ( 4 ) , nucleic acid constituents (5), neurotransmitters (6),and amino acid enantiomers (7). The present report describes a system that yields near base line resolution for dansylated methylamine and danyslated methyl-d3-amine. The system uses a phosphateborate buffer system containing 25 mM sodium dodecyl sulfate (SDS)and 20% methanol (MeOH). The two labeled amines, shown in Figure 1, differ by only one trideuterated methyl group. 0003-2700/89/0361-0491$01.50/0
EXPERIMENTAL SECTION Materials. A fused silica capillary with dimensions 50 pm i.d. and 150 pm 0.d. was obtained from Polymicro Technologies (Phoenix, AZ). Electrophoresis grade SDS was obtained from Bethesda Research Labs (Gaithersburg, MD). Methylamine, methyl-d3-aminehydrochloride, and octylamine were obtained from Aldrich (Milwaukee,WI).Dansyl chloride and n-hexylamine were obtained from Sigma (St. Louis, MO). Dodecylamine was obtained from Eastman Kodak (Rochester,NY), and formamide was obtained from Fisher Scientific (Raleigh, NC). Deionized water was further purified with a Barnstead Nanopure system (Boston, MA). Equipment. Two detectors were used in this work. Dansylated compounds were detected by using a variable wavelength 0 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
a \
Table I. MECC Capacity Factors for Test Compounds" symbol to
/
tD
tl
C=i=O
t6
1
NH CH3
NH
t8
CD
tl2
formamide DNS-NHCD, DNS-NHCH3 DNS-hexylamine DNS-octylamine DNS-dodecylamine
tmc
Flgure 1. Structures of dansylated methylamine and dansylated
time, s 1816
4890 4930 -4748 -4576 -4519
R, 0 0.813
0.820 71.3 279 9430
-4517
Conditions given in text.
methyl-d,-amine. fluorescence detector (8) with an excitation wavelength of 365 nm and a 470 nm cut on emission filter. To obtain tomeasurements, the same capillary was placed in a W detector (9) operated a t 229 nm. All injections were performed with an autoinjector described previously (IO). A 2-Hz RC low-pass filter was used on all fluorescence data. A time constant of 0.1 s was wed on absorbance data. A 30-kV dc power supply obtained from Spellman High Voltage Electronics Corp. (Plainview, NY) was used. Methods. Fused silica capillaries were cut to a length of 120 cm and a window was burned through the polyimide coating 15 cm from one end. Fresh capillaries were treated with the following rinses adapted from the work of Lauer and McManigill(11): 10 min with 1N KOH, 10 min with 0.1 N KOH, 10 min with water, and 20 min with the operating buffer. The electrophoretic buffer used was similar to those reported previously (I,4,12). A stock solution of sodium phosphate-borate buffer containing SDS was mixed with methanol, sodium bicarbonate, and deionized water tQ give final concentrations of 20% MeOH (v/v), 25 mM SDS, 25 mM Na2HP04,0.625 mM sodium borate, and 4.28 mM sodium bicarbonate. The pH was adjusted to 8.0 with NaOH. Dansylation of the amines was accomplished by first mixing M solution of dansyl chloride in acetonitrile at a a 1.4 X M methyl-d,-amine hydrovolume ratio of 1:6 with 1.4 X chloride in a 0.05 M sodium bicarbonate buffer at pH 9.5 or with 1.4 X M methylamine in a 0.05 M sodium bicarbonate buffer a t pH 9.5. A blank solution was made by mixing the dansyl chloride solution 1:6 with the bicarbonate buffer containing no amine. The solutions were gently shaken and allowed to react at room temperature in the dark for 20 min. Thirty-microliter portions of the resulting solutions of tagged amine were diluted to a total volume of 3 mL with buffer similar to the electrophoresis buffer but without additional sodium bicarbonate. The resulting solution is 2.0 X l@M in tagged amine (assuming all of the amine present reacts). Since the deuterated amine is in the form of a hydrochloride, and this changes the reaction pH slighty, the tagging method is less efficient for the deuterated compound when compared to the protiated compound. Thus, to produce relatively even peak heights for the two amines, the final sample mixture was made by mixing 2 part9 of the resulting DNS-NHCD3 solution to 1 part of the DNS-NHCH3 solution. Dansyl derivatives of hexylamine, octylamine, and dodecylaminewere made in a similar manner. The elution time of solute completely excluded from the micelles, to, was measured in two ways. First it was measured by the refractive index change a t 229 nm due to an injection of operating buffer containing 0% MeOH. While the base-line deflection was rather small, it was repeatable. This value of to was confirmed with an injection of formamide which gives a strong absorption peak at this wavelength. The elution time of solute completely included in the micelles, t,, was determined from the migration behavior of a series of homologous dansyl amines, as will be discussed. All injections were hydrostatic (IO) and involved raising one end of the capillary 10 cm in height for 8 s. Following the injections, voltages of 5,10, and 15 kV were applied for 1min each before applying the final run voltage of 30 kV. Data aquisition was begun with the application of 30 kV; therefore, unless otherwise noted, all migration times reported here have been corrected for this step voltage application procedure by adding 60 s to the measure migration times. A ramped voltage application has been previously reported by McCormick (13). He observed that a
compound
ramped application of the run voltage following an injection improved separation efficiencies. This was also observed by Sally Swedberg and John Christianson of HewlettPackard with regards to a stepped voltage application. Their suggested explanation is that if the run voltage causes heating of the buffer, as the buffer expands it will cause a portion of the sample slug to back up into the buffer reservoir. A stepped or ramped voltage application allows the sample to migrate a short distance into the capillary before any significant buffer heating and expansion occurs (14).
RESULTS AND DISCUSSION Capacity factors can be determined by using eq 1 (1)where t o is the migration time of solute totally excluded from the micelles, t,, is the migration time of solute completely included in the micelles, and tR is the migration time of the solute of interest. Since the formamide migration time was
the same as the methanol migration time, but was more easily observed due to its strong absorbance, formamide is assumed not to enter the micelles and the migration time of formamide was used as to. The corrected t o value is listed in Table I. To obtain t,,, dansylated methylamine, dansylated hexylamine, dansylated octylamine, and dansylated dodecylamine were used. As the carbon chain length increases, the compound increasingly favors partitioning into the micelles. Because of the added methanol, and hence decreased electroosmotic flow (121, the micelles were found to be migrating from negative electrode toward positive electrode. In order to measure the migration of the dansylated long chain amines, the same capillary was used but instead of applying +30 000 V as when finding to,tl, and tD,-30000 V was applied. The migration times of the dansylated long chain amines are listed in Table I. The minus sign indicates migration was from negative electrode to positive electrode. Since the dodecylamine favors the micelle more than the shorter amines, its migration is faster than the octylamine and hexylamine. Since the carbon number has increased by 4 over octylamine, but the migration time has decreased by less than 1 min, the dansylated dodecylamine is assumed to be migrating with a velocity very close to that of the micelle. T o see if this i_sthe case, t,, was assumed to be equal to tlz and the log of k'for tl, t 6 , and t8were plotted versus carbon number of the amines. The plot yields3 straight line with a correlation coefficient Of 0.9986 and a k'value of 9430 for dodecylamine. Using this k'value in eq 1 and solving for t,,, we obtain a t,, va;lue of -4517 s. When this value oft,, is used to recalculate k', and log k'versus carbon number is plotted again, a straight line results with a slope of 0.3601, an intercept of -0.4084, and a correlation coefficient of 0.9992. The dansylated dodecylamine could have been used as a t,, marker in this experiment with an error of only 2 s. The values reported in Table I, however, were calculated from the extrapolated t,, value and not from t1P
Sudan I11 is reported elsewhere (1,4) as having a migration time equal to t,,, but we could not detect a Sudan I11 peak a t the wavelengths our systems were operated. 9-Methyl-
ANALYTICAL CHEMISTRY, VOL. 61, NO. 5. MARCH 1, 1989
80
TIME (min)
83
Figure 2. Expansion of electropherogram of DNS-NHCD3 (peak A) and DNS-NHCH, (peak B) mixture. Conditions are as follows: 25 mM SDS, 2 0 % MeOH, 25 mM Na,HPO,, 0.625 mM Na2B,0,.10H,0, 4.28 mM NaHC03, pH 8.0; capillary, 50 pm i.d., 150 pm o.d., 120 cm (105 cm to detector); applied voltage, 30 kV; current, 25 pA; excitation wavelength, 365 nm; cut on emission wavelength, 470 nm.
anthracene was also used to try to determine t,,, but its migration time was -5280 s over a distance of only 15 cm. This would have been -39 960 s had it been migrated over the entire 105 cm capillary length. Since its migration time is much longer from negative electrode to positive electrode than the dansylated dodecylamine, 9-methylanthracene spends a considerable amount of time in the mobile phase and cannot be used to determine t,, in this system. We believe this to be the first MECC study to find t,, by the method presented here and suggest that it is a more reliable method than using a single compound as is usually done. This is especially true when organic solvents are added to the mobile phase. While it may be accurate to say that Sudan 111,for instance, is insoluble in aqueous buffer and will therefore be completely included in the micelles, it is questionable whether this is true in systems such as ours that employ 20% organic solvent. Figure 2 shows an expanded section of a separation of the two tagged methylamines. Peak A is DNS-NHCD3 and peak B-is DNS-NHCH3. The peak identities were confirmed by spiking the sample with additional DNS-NHCD3. Near base line resolution is obtained in less than 90 min. Table I lists corrected migration times and k ’values. With the k’values an CY (separation factor) of 1.009 is obtained for DNS-NHCD3 and DNS-NHCH3, where (Y is defined as
a ’1 / a
’ J ) .
Our system differs from other MECC systems in several ways. First of all, longer columns, 120 cm in length, were necessary to achieve the resolution required to separate these two compounds. The longer columns allow for use of higher voltages, although we still applied less than 1 W/m, which is reported elsewhere (15) as the useful limit in terms of heating effects. More importantly, however, our system uses 20% methanol, which is added to the mobile phase. The methanol is required because electrophoresis of the two dansylated amines in a micellar system without methanol results in no resolution (results not shown). As the concen-
493
tration of MeOH in the mobile phase is increased, DNS-NHCDBbegins to show a slightly more favorable affinity for the increasingly nonpolar mobile phase than does DNS-NHCHB. Organic-mobile phase additives, in addition to strongly reducing k’, also strongly reduce the electroosmotic flow which in turn increases the elution time and elution range (12). Methylamine and methyl-d3-amine have been separated before when both were tagged with fluorescein isothiocyanate (FITC) (16). In that case they were separated in less than 100 min by reverse-phase chromatography on a 5-pm open tubular liquid chromatography column utilizing laser-induced fluorescence detection. Capacity factors of 0.80 for the deuterated compound and 0.82 for the nondeuterated compound and an CY of 1.025 were reported. As in the present study, the deuterated analogue was found to be more hydrophilic than the protiated compound. It is interesting to note that the analysis time and resolution are comparable in the two studies. The two cases are not directly comparable, however, due to the different fluorescent tags used. Since FITC derivatives are negatively charged and the negative charge might interfere with the compound entering the negatively charged micelle, neutral dansyl chloride derivatives were used in the present study. We have demonstrated that MECC is a very powerful separating technique capable of resolving deuterated from protiated compounds. Addition of methanol to the mobile phase and use of long capillaries, high voltages, and a stepped application of the voltage all contribute to the large plate numbers necessary to perform this separation. In addition, we have suggested a new method for determining t,,, one that should be particularly useful in systems employing organically modified mobile phases.
LITERATURE CITED Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A,; Ando, T. Anal. Chem. 1984, 5 6 , 111-113. Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985. 57, 834-841. Otsuka, K.; Terabe, S.; Ando, T. J . Chmmatogr. 1985, 332, 219-226. Burton, D. E.; Sepaniak, M. J.; Maskarinec, M. P. J . Chromatogr. Sci. 1988, 2 4 , 347-350. Row, K. H.;Grelst, W. H.; Maskarinec, M. P. J . Chromatogr. 1987, 409, 193-203. Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1988, 60, 258-263. Gozel, P.; Gassman, E.; Mlchelsen, H.; Zare, R. N. Anal. Chem. 1987, 5 9 , 44-49. Green, J. S.;Jorgenson, J. W. J . Chromatogr. 1988, 352, 337-343. Walbrcehl, Y.; Jorgenson, J. W. J . Chromtogr. 1984, 375,135-143. Rose, D. J.; Jorgenson, J. W. Anal. Chem. 1988, 60, 642-648. Lauer, H. H.; McManiglll, D. Anal. Chem. 1988, 58. 166-170. Balchunas, A. t.; Sepaniak, M. J. Anal. Chem. 1988, 60, 617-821. McCormick, R. Anal. Chem. 1988, 6 0 , 2322-2328. Swedberg, S.; Christianson, J., Hewlett-Packard Corp., Palo Alto, CA, personal communication, 1988. Sepaniak, M. J.; Cole, R. 0. Anal. Chem. 1987, 59, 472-476. Dluzneski, P. R.; Jorgenson, J. W. HRC CC, J . Hlgh Resolut. Chromatogr Chromatogr. Commun. 1988, 7 7 , 332-336.
.
Michelle M. Bushey James W. Jorgenson* -
Department of Chemistry University of North Carolina Chapel Hill, North Carolina 27599-3290
RECEIVED for review July 25, 1988. Accepted November 15, 1988. Support for this work was provided by the National Science Foundation under Grant CHE-8607899 and by the Hewlett-Packard Corporation.
