Anal. Chem. 19S1, 63,109-114
109
Determination of Bioactive Peptides Using Capillary Zone Electrophoresis/Mass Spectrometry M. A. Moseley Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709, and Department of Chemistry, University of N o r t h Carolina, C B 3290, Chapel Hill, North Carolina 27514
L. J. Deterding and K. B. Tomer* Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709 J. W. Jorgenson Department of Chemistry, University of North Carolina, C B 3290, Chapel Hill, North Carolina 27514
Mlxtures of bloactlve peptides have been analyzed by capillary zone eiectrophoresls/mass spectrometry (CZE/MS) using an on-llne coaxial contlnuous-flow fast atom bombardment Interface. High separatlon efficiencies (up to 410 000 theoretkal plates) were obtained from low femtomole levels of peptkles. The analysis of baslc peptMes was accompifshed by ushg amlnopropykilylated CZE columns to mlnlmlze zone broadenlng due to adsorption effects. CZE/MS/MS data were acquired from femtomoie levels of peptldes In electrophoretic real tlme.
INTRODUCTION The combination of liquid chromatography (LC) with mass spectrometry (MS) has proven to be a very powerful tool for the analysis of peptide mixtures (1-11). The combination of capillary zone electrophoresis (CZE) with MS has rapidly gained in popularity due, in large part, to the clearly superior separation efficiency of CZE over LC, particularly in terms of efficiency per unit time. Separations in CZE are based upon the differential rate of migration of ionic species in an electric field, and CZE has proven to be capable of generating in excess of 1OOOOOO theoretical plates in less than 20 min (12-15) and greater than 100OOO plates in less than 1min (16). Thus, CZE separation efficiencies are 1-3 orders of magnitude higher than conventional LC. In addition, analysis times in CZE are short, typically 10-30 min, due to the high electric field strengths used with CZE (200-400 V/cm). Another feature that has made CZE an attractive biochemical separation technique is that peptides and proteins separated by CZE have been found to retain their biological activity (17). In addition to the high separation efficiencies associated with CZE, the low volumetric column flow rates (typically < 1 rL/min) minimize vacuum problems in interfacing CZE with MS. Capillary zone electrophoresis was first coupled with mass spectrometry by Smith et al. in 1987 (18),using an electrospray ionization (ESI) source a t atmospheric pressure with a quadrupole mass spectrometer, an approach which has proven to be quite useful (19-22) for the analysis of peptides. Electrospray ionization was first reported by Dole et al. (23, 24) and further developed by Fenn, who first reported the coupling of LC with MS using ESI (25). The reports by Fenn of the formation of multiply charged ions using ESI (26,27) has attracted widespread attention within the mass spec-
* To whom correspondence should be sent.
trometry community, and this ability of ESI to form multiply charged ions has been exploited for the MS analysis of proteins up to a mass of 133000 Da (21). A variant of electrospray known as ion spray has also been used to couple CZE with MS (28-30). All reports of CZE/ESI/MS to date have used quadrupole mass spectrometers equipped with cryogenic pumping systems. Research into coupling ESI with sector instruments is underway, but such work appears to be in preliminary stages (31). An alternate ionization technique that is more commonly used for the mass spectral analysis of peptides is fast atom bombardment (FAB). The technique, introduced in 1981 by Barber et al. (32), provides a powerful desorption/ionization source for the mass spectrometric analysis of polar, nonvolatile, and/or thermally labile analytes and has become the method of choice for the MS analysis of biopolymers. The coupling of FAB/MS with flowing liquid streams, developed by Ito et al. as Frit-FAB/MS (33) and Caprioli et al. as continuous-flow FAB/MS (34) has provided a potent interface for coupling LC with MS (2, 5 , 10, 11, 35-43). In 1988, our laboratories began the development of a CFFAB probe that was designed to be compatible with our nanoscale LC systems (both open tubular liquid chromatography and nanoscale packed-capillary LC)-coaxial CF-FAB (CF = continuous flow) (10, 38,43). These nanoscale LC systems place rigid requirements on the design of the interface since they utilize column mobile-phase flow rates of less than 100 nL/min, with injection and detection volumes of less than 20 nL. The previously reported LC/CF-FAB/MS interfaces were designed to operate with larger packed LC columns (4.6-0.22 mm i.d.), with mobile-phase flow rates (delivered to the mass spectrometer) of 3-10 pL/min. In these systems the matrix (typically glycerol) required for the FAB process was introduced either precolumn into the mobile phase and analyte solutions or postcolumn into the LC effluent by using a mixing tee. These approaches are not compatible with our nanoscale LC separation systems because the flow rate of the FAB matrix required for a stable FAB ion beam was greater than the total mobile-phase flow rates. Postcolumn introduction of the FAB matrix into the LC effluent is problematic for the same reason-postcolumn addition of the FAB matrix leads to severe chromatographic peak broadening. Therefore, an interface was designed which uses a coaxial flow of the LC effluent and FAB matrix in two separate fused-silica capillary columns. In this interface chromatographic band broadening was minimized because the mixing of the matrix and the LC effluent takes place only at the FAB
0003-2700/91/0363-0109$02.50/00 1991 American Chemical Society
110
ANALYTICAL CHEMISTRY, VOL. 63, NO. 2, JANUARY 15, 1991
FAB
1
Polypropylene Tubing I
1116’Tee
i Sheath Capillary
I
i Plexlglass Handle
Thumbscrew
i
Stainless Steel FA0 Probe Tip
I
Stamless Steel Probe Shah
Figure 1. Schematic of on-line capillary zone electrophoresis/coaxial continuous-flow fast atom bombardmentlmass spectrometry probe. Figure is not drawn to scale.
probe tip. The FAB matrix flow and LC flow are independently delivered to the FAB probe tip, allowing the composition and flow rate of the LC mobile phase to be optimized for the separation of the sample components and the composition and flow rate of the FAB matrix to be optimized for analyte detection. The success of the coaxial CF-FAB/MS system with our nanoscale LC systems and the similarities in column flow rates and injection and detection volumes between nanoscale LC and CZE led to the modification of the LC/coaxial CFFAB/MS probe for use with CZE/MS. Several modifications in the design were required for use with CZE due to the high voltages (30-50 kV) used with CZE. The resultant on-line coaxial continuous-flow CZE/FAB/MS system has proven to be capable of separating peptide mixtures with efficiencies of hundreds of thousands of theoretical plates, with MS detection limits of less than 10 fmol ( I I , 4 4 ) . System performance has been enhanced by several design modifications which have been made since the initial report. These modifications are here reported, along with the application of the CZE/MS system to the MS and MS/MS analysis of bioactive peptides.
EXPERIMENTAL SECTION Instrumentation. The FAB probe used in this CZE/MS work was fabricated in-house (Figure 1)and has essentially the same dimensions as the commercial VG continuous-flow FAB probe. This probe offers several advantages over commercially available continuous-flowFAB probes designed for use with LC. Whereas LC/CF-FAB/MS probes have vacuum seals external to the ion source, our CZE probe was designed such that the vacuum seal was made between the stainless steel probe shaft and the polyimide insulator at the tip of the probe. This permits the installation of a fused-silica helium cooling gas line (250 pm i.d., 350 pm 0.d.) into the probe shaft, terminating at the polyimide insulator. This allows the probe shaft to be constantly purged with helium to assist in the removal of the Joule heat generated within the CZE capillary. Failure to remove this Joule heat leads to the formation of radial temperature profiles in the CZE column, which causes zone broadening and, thus, a loss of CZE separation efficiency. We have found that the flow of FAB matrix in the sheath column effectively removes a significant portion of the Joule heat. Thus, He cooling may not be absolutely necessary. Exposure to air without cooling of the half of the column between the buffer and the sheath column has not led to observable adverse effects. The i.d. of the hole in the stainless steel FAB probe tip was carefully matched to the 0.d. of the sheath capillary column to prevent back-flow of the column effluent into the probe tip. This has been found to be necessary both to minimize zone broadening and to minimize ion source pressure fluctuations. For electrical insulation from the analyzer unit, the 1/16-in.stainless steel tee used to mate the two coaxial capillary columns was mounted in a Plexiglas handle on the probe shaft. The stainless steel probe shaft was lined with polypropylene tubing (1.2 mm i.d., 1.9 mm 0.d.) to provide additional electrical insulation between the probe shaft and the fused-silica capillaries. Failure to prevent contact between the CZE column and any grounded metal surfaces has been found to result in an immediate electrical short through the wall of the CZE column, such that an “electrodrilled” hole was formed in the capillary wall.
The CZE columns used in this interface were prepared from 12-15 pm i.d., 150 pm o.d., fused-silica capillaries (Polymicro Technologies, Phoenix, AZ). With the exception of the silylated CZE columns, all CZE capillary columns were pretreated by the method of Lauer and McManigill(45). While most CZE analyses are performed with column inner diameters ranging from 25 to 100 pm, the use of smaller i.d. CZE columns in the MS interface was necessary to minimize the vacuum-induced flow through the column. Vacuum-induced flow generates a parabolic flow profile within the CZE column, which, unlike the “plug” shaped profile resulting from electroosmotic flow (46),leads to electrophoretic zone broadening and a decrease in separation efficiency. The FAB matrix was pumped through the sheath capillary (160 pm i.d., 350 pm 0.d.) by using a syringe pump (Model pLC-500, Isco, Inc., Lincoln, NE). An in-line 0.5-pm frit filter (Supelco Inc., Bellefonte, PA) was used to filter the FAB matrix from the syringe pump to prevent pump seal particulates from entering the CZE/MS interface. In order to provide adequate back-pressure for regulation of the syringe pump flow, a 25 pm X 3 m fused-silica capillary column was used to connect the in-line filter on the syringe pump with the coaxial tee. The FAB matrix was 25% glycerol in water, modified with either heptafluorobutyric acid (pH 3.5) or ammonium hydroxide (pH 9). These modifiers increase conductivity of the FAB matrix, facilitating electrical contact between the end of the FAB probe tip and the end of the CZE column. This is required because the h8-kV FAB tip was used to complete the electrical circuit of the CZE column. The choice of modifier was based upon detection by the mass spectrometer as either positive or negative ions. It should be noted that the modification of FAB matrix pH allows the CZE separation of analytes as negative ions (basic CZE buffer) and their MS detection as positive ions (acidic FAB matrix) or vice versa. The FAB matrix flow rate was maintained at approximately 0.3 pL/min. A 60-kV reversible polarity power supply (Glassman High Voltage, Princeton, NJ) was used for electrophoresis. A highvoltage relay (Model H-25, Kilovac High Voltage, Santa Barbara, CA) was used to make electrical connections between the power supply and the CZE electrode. This relay was wired into safety interlock switches mounted on the sample/buffer box. The circuit was designed to force the potential of the CZE electrode to ground when the lid of the sample/ buffer box was opened. Incorporation of a timer circuit in the relay system permits the use of highvoltage switching for electromigration sample introductions. A potential drop of 30 kV was maintained across the 1-m CZE columns during the analyses, giving an electric field strength of 300 V/cm. Electromigration sample introductions were used in all analyses, typically at 150 V/cm for 2-5 s, resulting in “injections” of 0.1-0.6 nL. All analytes were prepared in 18-MQwater and were filtered (0.2 pm) immediately prior to analysis. On-column detection of analytes using UV absorption at 200 nm was accomplished by using a Model 500 variable wavelength detector (Scientific Systems Inc., State College, PA). CZE/UV data acquisition and processing were performed by using a 12-bit A/D converter (Scientific Solutions, Solon, OH) configured on an IBM PS2/30 computer (Research Triangle Park, NC). Processing of the CZE/UV data was performed with software written in-house at UNC. Buffer Systems. While a wide variety of buffers are used in CZE, volatile buffers have been found to be required for the stable operation of the FAB/MS system (44). The buffers used in this work were either 0.005 M ammonium acetate or 0.01 M acetic acid, with pH adjustments made with ammonium hydroxide. All buffers were filtered (0.2 pm) and degassed immediately prior to use. Preparation of APS Column. Aminopropyl-silylated ( U S ) fused-silica CZE columns were prepared from a modification of the method of Lukacs (47).Helium pressure (15 psi for 75 pm i.d. columns, 1500 psi for 13 pm i.d. columns) was used to pump the solutions (filtered, 0.2 pm) through the capillary. An acid treatment of the column was used to open silyl ether linkages to produce additional silanols for silylation. While fused-silicaAPS columns can be prepared without this acid treatment, the resulting columns were less durable. Acid treatment was accomplished by rinsing the column with a 6 M solution of HCl for approximately
ANALYTICAL CHEMISTRY, VOL. 63, NO. 2, JANUARY 15, 1991 4 h. The column was then dehydrated by installing it in a capillary GC (gas chromatographic) oven, purging with helium gas, and maintaining a temperature of 300 O C for 12-15 h. The column was then rinsed with toluene for approximately 10 min. Next, a 5 % solution of (3-aminopropy1)trimethoxysilane in toluene was pumped through the column for 2 h, followed by a 30-min rinse with toluene. The column was dried by pumping helium through the column for approximately 30 min. Prior to use, the column was flushed with the CZE buffer solution for approximately 1 h. Mass Spectrometer. A VG ZAB 4F mass spectrometer (VG Analytical, Altrincham, England) was used for this work. This instrument has been previously described in detail (48). The mass spectrometer is of B I E I- EzBz geometry, and is equipped with an Ion Tech fast atom bombardment gun. Xenon was used as the FAB gas (8 kV at 1 mA). Analyte ions were accelerated to 8 kV. The FAB ion source temperature was heated to 40 "C. The MS work utilized only the first two sectors (B,E,) of the instrument, and ions were detected in the third field-free region with a photomultiplier tube detector. The magnet was exponentially scanned at a rate of 3 s/decade over a mass range from 20 Da above the mass of the highest molecular ion species of interest to 20 Da below the mass of the lowest molecular ion species of interest. The MS/MS analyses utilized the first two sectors to focus the parent ions into the collision cell in the third field-free region where collisions with helium gas (50% parent ion beam attenuation) induced dissociation to daughter ions. The daughter ions were analyzed by using a 10 s/decade linked scan at constant B2/Ez,with detection of daughter ions in the fifth field-free region with a photomultiplier tube detector. Spectra are the result of a single scan. Data analysis was accomplished with a VG 11-250 data system. The results of the CZE/MS analysis have been output as single-ion electropherograms, plotting the intensity of the molecular ion species (protonated for positive-ion FAB/MS or deprotonated for negative-ion FAB/MS) as a function of time. Chemicals. All peptides and the glycerol were obtained from Sigma Corp. (St. Louis, MO) and were used as delivered. All solutions were prepared with 18-MO water (Milli-Q System, Millipore Corp., Bedford, MA). All other chemicals were obtained from Aldrich (Milwaukee, WI) and were used as delivered.
111
e
lee
I
I
Y.,?"
Time (minsec)
Figure 2. Single-ion electropherograms of the protonated molecular ions of a mixture of chemotactic peptides. (Numbers associated with peaks are scan numbers.) Note that the peptides were separated as negative ions (CZE buffer pH of 8.5) and were detected as poskive ions (FAB matrix pH of 3.5).
Time (rninsec)
B
(M-H)488
188
I
1
RESULTS AND DISCUSSION CZE/MS Analysis of Chemotactic Peptides. Chemotactic peptides are peptides which serve as cellular chemoattractants, producing concentration gradients of cells such as fibroblasts and monocytes (49). The chemotactic peptides chosen include several with only minor structural differences, which should present a challenging separation. The separation of these chemotactic peptides as negative ions, while detecting them as either positive or negative ions, illustrates a significant advantage of this coaxial interface. That is, the interface permits optimization of the composition and flow rate of the CZE buffer for the separation, while independantly optimizing the composition and flow rate of the FAB matrix solution for MS detection. Positive-Ion Detection. The chemotactic tripeptide MLF (Met-Leu-Phe) has been observed to give the lowest limits of detection by positive-ion FAB/MS of any peptide we have studied (500 am01 detected by using LC/coaxial CF-FAB/MS (IO)). The separation of a mixture of chemotatic peptides is shown in Figure 2. This mixture, composed of VGVAPG (Val-Gly-Val-Ala-Pro-Gly) (2.5 X M), MLF (2.5 X lo-, M), N-formyl MLF M), N-acetyl MLF M), Nformyl AGSE (Ala-Gly-Ser-Glu) (lo4 M), oxidized MLF, and oxidized N-acetyl MLF, was separated as negative ions by using a pH 8.5 CZE buffer and detected as positive ions by using a pH 3.5 FAB matrix solution. Separation efficiencies for this analysis ranged from 420 000 to 34 000 theoretical plates. [Theoretical plates, N, = 5.54(migration time/peak width a t half-height)2(50).]Good signal to noise ratios (S:N) were observed for all peaks, with the exception of N-acetyl MLF. The (M + H)+ ion of N-acetyl MLF was obscured by
Figure 3. (A) Single-ion electropherograms of the deprotonated molecular ions of a mixture of chemotactic peptides. (Numbers associated with peaks are scan numbers.) Note that the peptides were separated as negative ions (CZE buffer pH of 8.5) and were detected as negative ions (FAB matrix pH of 9). (B) Spectrum of MLF (background subtracted) obtained from this separation.
+
the [(glycerol), H]+ ion cluster. The amounts of peptides analyzed ranged from 3.9 fmol of MLF (3.9 fmol total of MLF and oxidized MLF) to 17 fmol of N-formyl AGSE. The oxidized peptides were not deliberately added to the analyte mixture; rather they were the result of the partial degradation of the parent peptide. The observation of peaks with masses 16 Da above that of the parent peptides indicated the presence of oxidized species, and MS/MS analysis confirmed the addition of 16 Da to the methionine residue of the parent peptide. These results indicate the utility of CZE/MS for the analysis of peptide mixtures, for both impurity analysis (oxidized species) and the differentiation of structurally similar peptides (MLF, N-acetyl MLF, and N-formyl MLF). Negative-Ion Detection. This CZE/MS analysis of cheM) was performed by using motactic peptides (at 2.4 X pH 8 CZE buffer and pH 9 FAB matrix; thus the peptides were both separated and detected as negative ions (Figure 3). The sensitivity of the FAB/MS system for the detection of MLF and N-formyl MLF as negative ions is approximately
112
ANALYTICAL CHEMISTRY, VOL. 63, NO. 2, JANUARY 15, 1991 m
N
=
A '
3 555 480 000
1
Bradykinin ,!I
i
1
1 q{
I
E P 4
504 100
m L 522 N =420000 ' 5 lemlomoles
I1
Proclolln n L 649 N - 360 000
6.0
60 'emlomoles
Trne :min i
18.6
Time (min:sec)
Figure 4. Single-ion electropherograms of the protonated molecular ions of a mixture of neuropeptides. (Numbers associated with peaks
are scan numbers.) The peptides were separated as negative ions (CZE buffer pH of 8.5)and were detected as positive ions (FA6 matrix pH of 3.5). 1 order of magnitude less than that under positive ion conditions. In contrast to the detection of these analytes as positive ions, N-acetyl MLF can be detected under negative-ion conditions due to the decreased abundance of the glycerol oligomer. The detection limits of VGVAPG and N-formyl AGSE and the oxidized species (S:N > 3:l) are significantly higher, such that these compounds are not observed in Figure 3A. For the three tripeptides, good signal to noise ratios were obtained in the negative-ion mode with analyte levels less than 100 fmol. The spectrum (matrix subtracted) of MLF obtained from this analysis is shown in Figure 3B. CZE/MS Analysis of Neuropeptides. Neuropeptides have been of particular interest to our research groups, and a variety of neuropeptides have been successfully analyzed by using the CZE/MS system. For example, the analysis of a mixture of four neuropeptides (Figure 4) demonstrates the ability of the CZE/FAB/MS system to separate small amounts of neuropeptides [60-75 fmol/component (1.5-2.0 X M)] with efficiencies ranging from 220 000 to 480 000 theoretical plates. Ammonium acetate buffer at pH 8.5 was used for this analysis. Three of these neuropeptides [leucine-enkephalinamide (YGGFL-NH,), morphiceptin (YPFP-NHJ, and FLEEI (Phe-Leu-Glu-Glu-Ile)]are composed of neutral or acidic amino acids, while proctolin (RYLPT) has a single basic arginine residue. Thus, a t the basic p H of the CZE buffer used in the analysis, leucine-enkephalin, morphiceptin, and FLEEI have a net negative charge, while proctlin is effectively neutral in net charge. On the basis of size and charge, proctolin would be expected to be the first peptide to migrate from the CZE column, however, proctolin was the third. This change in the migration order may be correlated with reversible adsorption of proctolin onto the capillary walls. Bare fused-silica in contact with aqueous solutions with a pH >1 will have a negatively charged wall due to the deprotonation of surface silanols. This induces an electrostatic repulsion between the negatively charged walls of the bare fused-silica CZE column and the negatively charged peptides, and, thus, they will not adsorb onto the walls of the capillary column. Proctolin, however, has a positively charged arginine residue, and the interaction of this positively charged residue with the negatively charged capillary walls is manifested by a retardation in its migration rate. Analysis of Basic Peptides. Most separations of peptides by CZE using bare fused-silica columns are performed with basic buffer solutions (pH of buffer > p l of peptides) in order to induce an electrostatic repulsion between the column walls and the peptides (45). This approach to the analysis of basic
B
~
Bradykinin ! I
Figure 5 . CZE separation of two basic neuropeptides, detected by UV absorption at 200 nm: (A) separation with bare fused silica CZE column; (B) separation with APS-treated CZE column.
peptides has been limited by the high pK, values for lysine and arginine (10.5 and 12.5, respectively), which require extraordinarily high pH values for the removal of their positive charge. Alternate approaches to the analysis of basic peptides, as well as proteins, include the addition of potassium ions to the CZE buffer (51),the use of low pH phosphate buffers in surface-modified silica capillaries (52),coating the column wall with methylcellulose (53), and silane modification of the column wall (15, 53). The extent of this adsorption problem can be observed in the CZE/UV analysis of a solution of two basic peptidesbradykinin ( 2 arginine residues) [RPPGFSPFR] and lysbradykinin ( 2 arginine and one lysine residue) [KRPPGFSPFR]. The analysis of this mixture with a pH 7.5 buffer (typically used in our CZE/MS analyses) in bare fused silica (Figure 5A) results in severe, deleterious zone broadening resulting from the adsorption of the positively charged peptides onto the wall of the CZE column. Although these conditions are not optimum for this specific separation, they are typical of general conditions used for the CZE separation of unknown peptides and can, therefore, be considered representative of potential results. Optimum conditions (deprotonation of Lys and Arg residues) would necessitate an approximate buffer pH of 13, which is quite severe. Silylation of the CZE column with (3-aminopropy1)trimethoxysilane (APS) (47) results in a column wall where the surface silanols have been modified with aminopropyl groups. The pK, of n-propylamine is 3.4. Thus, at a CZE buffer pH of 3.4, the column walls will have a net positive charge, which will electrostatically repel the positively charged basic peptides. Analysis of the mixture of the basic peptides by CZE/UV using the APS column (Figure 5B) with a pH 3.4 buffer resulted in greatly improved peak shape and separation efficiency. It must be noted that the electroosmotic flow in these APS columns is reversed. flowing from the low-voltage elec-
ANALYTICAL CHEMISTRY, VOL. 63, NO. 2, JANUARY 15, 1991
0
4'
A.
5 65. u6l
=
3 a0
Bradykinin
55: !4. 45. 41.
mi2 1061 N = 410,000 55 lemlomoles
'2'
1
113
Tyr Gly Gly Phe Met-NH,
Msis
m i 573
Lys-Bradykinin miz1189 N 350.000 46 lemlomoles i
.2 31.
I
zE
3. 25. 8. I6
Figure 6. CZE/MS analysis of the two basic neuropeptides,with data plotted as a summed-ion electropherogram of the protonatedmolecular ions. (Numbers associated with peaks are scan numbers.) The peptides were separated as positive ions (CZE buffer pH of 3.4) and were detected as positive ions (FAB matrix pH of 3.5).
): 601
Mel-Enke~tgnamide 21 lemtomoles
40 28 8
88. (8
28.
IOrJ
1
Phe-Leu-Glu-Glu-lle MS/MS or miz 650
(M+H)'
n
50
0
mlz Figure 8. CZEIMSIMS analysis of the three neuropeptides whose separation is shown in Figure 7. The peptides were separated as negative ions (CZE buffer pH of 8.5) and desorbed, fragmented, and detected as positive ions (FAB matrix pH of 3.5).
mi2 650 37 lemtomoles
FLEEI. The CZE analysis was then repeated three times with a M solution (injecting 420-840 fmol of the analytes), acquiring MS/MS data from each analyte as they migrated from the CZE column. The data acquisition software of the mass spectrometer required three separate runs to acquire MS/MS data from all three peptides. Under these conditions, severe overloading of the column, the analyte peaks overlapped. The MS/MS spectra of the peptides (Figure 8) clearly show daughter ion fragments resulting from fragmentation of the peptide backbone as well as amino acid side chain fragments. The nomenclature used for the peptide fragments is that developed by Roepstorff (54) as modified by Biemann (55). The MS/MS data obtained from methionine-enkephalin and methionine-enkephalinamide contain sufficient daughter ion fragments to allow the elucidation of the amino acid sequence, whereas the MS/MS data from FLEEI, while insufficient for complete sequence determination, is more than adequate for structure confirmation.
ACKNOWLEDGMENT The invaluable assistance of Bob Hall, Chief of Instrumentation, National Institute of Environmental Health Sciences, in the design and fabrication of the CZE/FAB/MS probes used in this work is gratefully acknowledged. Registry No. Val-Glu-Val-Ala-Pro-Gly, 92899-39-3; MetLeu-Phe, 59881-08-2;oxidized N-acetyl Met-Leu-Phe, 130523-71-6; oxidized Met-Leu-Phe, 130523-72-7; N-formyl Met-Leu-Phe, 59880-97-6; N-formyl Ala-Gly-Ser-Glu, 100929-80-4; N-acetyl Met-Leu-Phe, 73572-34-6; Phe-Leu-Glu-Glu-Ile, 62733-72-6; Tyr-Gly-Gly-Phe-Met-NH2,60117-17-1; Tyr-Gly-Gly-Phe-Met, 58569-55-4; Leu-enkephalinamide, 60117-24-0; morphiceptin, 74135-04-9; proctolin, 57966-42-4; Lys-bradykinin, 342-10-9; bradykinin, 58-82-2; met-enkephalinamide, 60117-17-1; metenkephalin, 58569-55-4.
LITERATURE CITED (1) Desiderio, D. M.; Fridland, G. H. J . Liquid Chromatcgr. 1984, 7 , 317. (2) Ashcroft, A. E. Org. Mass Spectrom. 1987, 22, 754. (3) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 5 9 , 2642. (4) Caprioli, R. M.; DaGue, 6.; Fan, T.; Moore, W. T. Biocbem. Siopbys. Res. Commun. 1987, 146, 291. (5) Caprioli, R. M.; Moore, W. T.; DaGue, 6.; Martin, M. J . Chromatogr. 1988, 443, 355. (6) Fridland, G. H.; Desiderio, D. M. J . Chromatogr. 1986, 379, 251. (7) Fridland, G. H.: Desiderio, D. M. Life Sci. 1987, 41, 809. (8) Hutchinson, D. W.; Woolfitt, A. R.; Ashcroft, A. E. Org. Mass Spectrom. 1987, 22, 304.
114
Anal. Chem. 1991, 63, 114-120
(9) Sakairi, M.; Kambara. H. Anal. Chem. 1988, 60, 774. ( 10) Moseley, M. A.; Deterding, L. J.; Tomer, K. B.; Kennedy, R. T.; Bragg,
(1I )
(12) (13) (14) (15) (16) (17) (18) (19)
N. L.; Jorgenson, J. W. Anal. Chem. 1989, 6 1 , 1577. Moseley, M. A.; Deterding, L. J.; Tomer, K . B.; Jorgenson, J. W. J. Chromatogr. 1989, 480, 197. Jorgenson, J. W.: Lukacs, K. D. J . Chromatogr. 1981, 218, 209. Jorgenson, J. W.; Lukacs. K. D. Clin. Chem. 1981, 27, 1551. Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298. Jorgenson, J. W.; Lukacs. K. D. Science 1983, 222, 266. Nickerson, B.; Jorgenson, J. W. HRC& CC, J. High Resolut Chromatog. Chromatogr. Commun. 1988, 1 1 , 553. Rose. D. J.; Jorgenson, J. W. J. Chromatogr. 1988, 438, 23. Olivares, J. A,; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987, 59,1230. Smith, R. D.: Barinaga, C. J.; Udseth. H. R. Anal. Chem. 1988, 60, 1948.
(21) Loo, J. A ; Udseth, H. R.; Smith, R. D. Anal. Biochem. 1989, 179, 404. (22) Smith, R. D.; Loo, J. A.; Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R. J. Chromatogr. 1989, 480, 211. (23) Dole, M.: Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J . Chem. Phys. 1968, 49, 2240. (24) Dole, M.; Cox, H.L., Jr.; Gieniec, J. Adv. Chem. Ser. 1973, 125, 73. (25) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985. 5 7 , 675. (26) Wong, S. F.; Meng, C. K.; Fenn, J. B. J . phys. Chem. 1988. 92, 546. (27) Fenn. J. B.; Mann, M.; Meng. C. K.; Wong, S. F.; Whitehouse. C. M. Science 1989, 246, 64. (26) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R. J. Chromatogr. 1988, 458, 313. Lee, E. D.; Henion, J. D.; Budde, W. L. Biomed. Environ. (29) Edlund. P. 0.: Mass Spectrom. 1989, 18, 233. (30) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R . Biomed. Environ. Mass Spectrom. 1989, 18, 253. (31) Allen, M. H.; Lewis, I.A. S. Rapid Commun. Mass Spectrom. 1989, 3. 255. (32) BaGer, M.; Bordoli. R. S.; Sedgwick, R. D.; Tyler, A. N. J . Chem. SOC., Chem. Commun. 1981, 325.
(33) Ito, Y.; Takeuchi, T.; Ishii, D.; Goto, M. J. Chromatogr. 1985, 346, 161. (34) Caprioli, R. M.; Fan, T.;Cottrell, J. S. Anal. Chem. 1988, 5 8 , 2949. (35) Caprioli, R. M.; DaGue, B. B.; Wilson, K. J. Chromatogr. Sci. 1988, 26, 640. (36) Chen, K . H.: Cotter, R. J. Rapid Commun. Mass Spectrom. 1988, 2 , 237. (37) Dasgupta, A.; Ayanoglu, E.; Tomer, K. B.; Djerassi, C. Chem. Phys. Lipids 1987, 43, 101. (38) dewit. J. S. M.; Deterding, L. J.; Moseley. M. A,; Tomer. K. B.; Jorgenson, J. W. Rapid Commun. Mass Spectrom. 1988, 2 , 100. (39) Games, D. E.;Pleasance, S.; Ramsey, E. D.; McDowall, M. A. Biomed. Environ. Mass Soectrom. 1988. 15. 179 (40) Lisek, C. A.; B&ley, J. E.; Benson,'L. M.; Yaksh, T. L.; Jardine, I . Rapid Commun. Mass Spectrom. 1989, 3 , 43. (41) Norwood. D. L.; Kodo, N.; Millington, D. S. Rapid Commun. Mass Spectrom. 1988, 2 , 269. (42) Vekey, K.; Edwards, D.; Zerilli, L. F. J. Chromatogr. 1989, 488, 73. (43) Deterding, L. J.; Moseley, M. A.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1989, 61, 2504. (44) Moseley, M. A.; Deterding, L. J.; Tomer, K. B.; Jorgenson, J. W. Rapid Commun. Mass Spectrom. 1989, 3 , 87. (45) Lauer, H. H.; McManigill, D. Anal. Chem. 1988, 58, 166. (46) Rice, C. L.; Whitehead, R. J . Phys. Chem. 1965, 69, 4017. (47) Lukacs. K. D.Diss. Abstr. Int. 8 1983, 44, 3766. (48) Cole, R. B.; Guenat, C. R.: Gaskell, S. J. Anal. Chem. 1987, 59, 1139. (49) Freer, R. J.; Day, A. R.; Muthukumaraswamy, N.; Pinon, D.; Wu, A,; Showell, H. J.; Becker, E. L. Biochemistry 1982, 2 1 , 257. ( 5 0 ) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography; John Wiley B Sons, Inc.: New York, 1979; p 31. (51) Green, J. S.;Jorgenson, J. W. J . Chromatogr. 1989, 478, 63. (52) McCormick, R. M. Anal. Chem. 1988, 60, 2322. (53) Hjerten, S.J. Chromatogr. 1985, 347, 191. (54) Roepstorff, P.; Fohlman, J. Biomed. Environ. Mass Spectrom. 1984, 1 1 , 601. (55) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99.
RECEIVED for review June 26,1990. Accepted October 8,1990.
Hydrocarbon Type Determination of Naphthas and Catalytically Reformed Products by Automated Multidimensional Gas Chromatography Julius J. Szakasits* and Robert E. Robinson Shell Development Company, Westhollow Research Center, Houston, Texas 77251-1380
A multidimensional gas chromatographlc system Is descrlbed for obtalnlng the content of paraffln, naphthene, and aromatlcs (PNA) accordlng to both grouptype separation and separation by the number of carbons In hydrocarbon mixtures. Several examples of measured PNA results for callbration standards and practical process/product streams are glven to Illustrate the accuracy, reproduclblllty, and repeatability of the method. The Importance of uslng purified hydrogen as the carrier gas and a speclally prepared molecular sieve 13X wall-coated capillary column for Improved PIN separatlon is stressed.
INTRODUCTION Within the petroleum-refining industry, the importance of detailed characterization of unit feedstocks and process/ product streams for obtaining the content of paraffin, naphthene, and aromatics has been long recognized. In addition to physical property data, knowledge of the different hydrocarbon type distributions and individual components for the many different refinery streams provides information that directly impacts monitoring unit operations, product
specifications, and the development of cost-effective integrated petroleum processes. In catalytic reforming, which is the main process for the production of aromatics (high octane reformates) in refinery operations, the ultimate objective of naphtha and reformate analysis is to provide quantitative information about the distribution of hydrocarbons among the different types as rapidly as possible in order to assist in plant surveillance and process tuning. The failure to recognize poor plant performance or upset a t an early stage can lead to serious loss in reformate yields and/or a premature catalyst regeneration. Naphtha feedstocks to catalytic reformers are complex hydrocarbon mixtures that boil within the distillation range of approximately 25-220 "C. These feedstocks are essentially olefin-free with typical chemical class compositions of 85-95 % by volume saturated aliphatics and 5-15% by volume aromatics (1). With reforming conversion, typical reformates contain 40-55% by volume saturated aliphatics and 45-60% by volume aromatics. The principal techniques employed for determining the content of paraffin, naphthene, and aromatics (PNA) in naphthas and catalytically reformed products are mass spectrometry (MS) ( 2 ) ,high-performance liquid chromatography (HPLC) (3, 41, and capillary column gas chro-
0003-2700/91/0363-0114$02.50/00 1991 American Chemical Society
