Anal. Chem. 1986, 58,9-14
values, but as shown by the points plotted as squares in the figure, all of these four points gave results that were within the limits of two standard errors of the mean of the regression line. ACKNOWLEDGMENT We thank Normal Gershfeld, NIADDK, for his encouragement and provocative discussions during the preparation of this manuscript. Registry No. HzO,7732-185; CH3CN,75-05-8; MeOH, 67-56-1; EtOH, 64-17-5; i-PrOH, 67-63-0; (CH3)&0, 67-64-1; NH,Ac, 631-61-8; AcO(CHz)2NMe3f,51-84-3; HCONHMe, 123-39-7; methoxyethanol, 32718-54-0;tetrahydrofuran, 109-99-9;dimethyl sulfoxide, 67-68-5; norepinephrine, 51-41-2. LITERATURE CITED (1) Liberato, D. J.; Fenselau, C. C.; Vestal, M. L.; Yergey, A. L. Anal. Chem. 1983, 55, 1741. (2) Yergey, A. L.; Liberato, D. J.; Miillngton, D. S. Anal. Blochem, 1984, 139, 278.
9
(3) Pilosof, D.; Kim, H. Y.; Dyckes, D. F.; Vestal, M. L. Anal. Chem. 1984, 56, 1236. (4) Covey, T.; Henlon, J. Anal. Chem. 1983, 55, 2275. (5) Park, M. F.; Liberato, D. J.; Yergey, A. L.; Folk, J. E. J . Biol. Chem. 1984, 259, 12123. (6) Liberato, D. J.; Yergey, A. L., unpublished results. (7) Liberato, D. J.; Weintraub, S.T.; Yeraey, _ .A. L. Biomed. Mass Saecfrop., in press. (8) Glajch, J. L.; Kirkland, J. J.; Squire, K. M.; Minor, J. M. J . Chromatogr.
---.
l Q 8 0 199 - - , 57. -
S.;Owen, B. B. "The Physical Chemistry of Electrolytic Solutions"; Reinhold: New York, 1943; p 33. Vestal, M. L. Mass Specfrom. Rev. 1983, 2, 447. Vestal, M. L. private communication. Yergey, A. L.; Llberato, D. J. "32nd Annual Conference on Mass Spectrometry and Allied Topics" (Proceedings), San antonio, TX, 1984; p 90. Iribarne, J. V; Thomson, E. A. J . Chem. Phys. 1976, 6 4 , 2287. Harned, H. S.;Owen, B. E. "The Physical chemistry of Electrolyte Solutions"; Relnhold: New York, 1943; p 543. Voyksner, R. D.; Eursey, J. T.; Pellizzarl, E. D. Anal. Chem. 1984, 56,
(9) Harned, H. (10) (11) (12) (13) (14) (15)
1507.
RECEIVED for review July 1,1985. Accepted August 27, 1985.
Phospholipid Molecular Species Analysis by Thermospray Liquid Chromatography/Mass Spectrometry Hee-Yong Kim a n d Norman Salem, Jr.*
Section of Analytical Chemistry, Laboratory of Clinical Studies, DICBR, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892
A technlque for rapld and detailed molecular species analysls of phospholiplds was developed by using thermospray liquid chromatography/mass spectrometry (LCIMS). Wlth a hlgh percentage of organlc solvent in the carrier solution and wlth the filament on, synthetic molecular specles of phosphatldylchollnes (PC) and phosphatldylethanolamlnes(PE) were detected as molecular ion specles along wlth slmple fragment Ions, which provide detalied Information concerning fatty acld composltlon and phosphate ester head group identlty. I n conjunction wlth on-llne reversed-phase chromatography, uslng a hexane-methanoi-0.1 M ammonium acetate mixture as the moblle phase, thls technlque was generalized for analysls of natural mixtures of PC or PE species. Even for a complex mixture such as egg phoaphatldylethanolamlne, reliable structural Information for each molecular specles was readlly obtalned in 15 mln. The present detectlon limit of thls technique is in the 10-100 ng range.
Phospholipids are complex mixtures of many molecular species. Phospholipid classes often have characteristic fatty acyl profiles (1,2) and the degree of unsaturation is one of the primary determinants of membrane physical properties (3). Therefore, techniques for analyzing the distribution of individual molecular species has been of great value in lipid and membrane research. Although a variety of techniques including argentation thin-layer (4-7), column (B), and gas chromatography (9) have long been employed for phospholipid molecular species analysis, these procedures entail multistep procedures that are often laborious and time-consuming. Rapid analysis by high-performance liquid chromatography (HPLC) his become more popular recently (10-16), but severe
limitations are imposed on solvent system selection since UV absorption of underivatized phospholipids only occurs near 200 nm, with a low extinction coefficient. Mass spectrometry (MS) has proven useful for phospholipid analysis since it provides structural information. However, derivatization is necessary for conventional GC/MS (17, 18), and direct analysis of intact phospholipid molecules has been a difficult task. Some soft ionization techniques such as chemical ionization (19,20), fast atom bombardment (FAB) (21), field desorption (FD) (22,231, and secondary ion mass spectrometry (SIMS) (24,25) have demonstrated the ability to produce molecular ion and partial structural information, but the extensive off-line procedures involved prevent the efficient analysis of complex mixtures. Recently, an on-line procedure for phospholipid class separation was reported using a moving b e l t 4 1 system (26). Thermospray liquid chromatography/mass spectrometry (LC/MS) has already shown great potential for the analysis of nonvolatile biological samples since the spectra of intact molecules can be readily produced without derivatization (27). This technique also allows on-line chromatographic operation at conventional flow rates. In the present study, we applied this technique to the analysis of synthetic and natural phospholipid molecular species. EXPERIMENTAL SECTION Synthetic and egg phospholipids were purchased from Avanti Polar Lipids, Inc. (Birmingham, AL), and were used without further purification. The separation of phospholipids was performed by using methanol-hexane-0.1 M ammonium acetate/ water as the solvent system with an Altex Ultrasphere-ODS column (3 Fm, 4.6 mm X 7.5 cm) (Berkeley, CA). Samples were dissolved in the mobile phase, injected into an Altex 210A injector with a 50-pL sample loop, and delivered into the LC/MS system by a Beckman Model 114M pump.
This article not subject to US. Copyrlght. Published 1985 by the American Chemical Society
10
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY
1986
The thermospray LC/MS system used a Vestec interface (Vestec,Houston, TX) mounted on an Extrel400-2 quadrupole mass spectrometer that was modified slightly to accommodate the thermospray interface. The optimum temperatures for the vaporizer and downstream jet in this phospholipid analysis were found to be 148 and 275 "C, respectively. Since mobile phase extremely high in organic content was necessary for dissolving and eluting phospholipids, sufficient ionization could not occur without an external ionizing source. Thus, the electron-emitting filament (0.2 mA) was turned on throughout the experiment. The ion source and analyzer housing were equipped with 360 L/s turbomolecular pumps (Leybold-Heraeus, Germany), which in turn were backed by standard mechanical pumps. Most of the liquid coming into the ion source was pumped away by an auxiliary mechanical pump, connected opposite to the thermospray vaporizer, through a dry iceacetone cold trap. The analyzer pressure was maintained at approximately 1.5 X torr with a mobile phase flow rate of 1 mL/min. An Extrel EL-1000 data system was used for data acquisition and mass spectrometer control.
RESULTS AND DISCUSSION Thermospray Spectra of Phospholipids. The positive ion spectra of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were obtained by the use of thermospray with the filament current on for more efficient ionization. A simple fragmentation pattern containing information about fatty acyl composition and phosphate ester head group identity was obtained along with molecular ions. The major fragments are similar to those of ammonia CI (19),but the thermospray technique produced much simpler spectra with extremely low background. Typical examples are shown for synthetic 16:0,18:1-PC (Figure la) and 16:0,18:1-PE (Figure Ib). Two micrograms of eac'i of these lipids was injected directly into the thermospray mass spectrometer using 9 5 5 methanol/O.l M ammonium acetate as carrier solvent at a flow rate of 1mL/min. As shown in Figure la, the base peak was the diglyceride ion at m / z 578, resulting from the loss of the phosphocholine head group. The ions derived from phosphocholine were detected a t m / z 142 and 184. The peak a t m / z 142 was usually more intense than that at m / z 184 due to the loss of trimethylamine from the ammonium adduct of phosphocholine as reported for ammonia CI mass spectrometry. This phenomenon can also occur with intact PC molecules as (M - 41)' ions were sometimes observed. Peaks at m/z 313 and 339 represent the glyceride ions containing 16:O fatty acid or 18:l fatty acid, respectively. They are believed to arise from the hydrolysis of either fatty acyl moiety from the diglyceride ion. The mass values of the glyceride ions permit easy deduction of fatty acyl composition of phospholipids. Since no preference in hydrolysis of either fatty acyl group was observed, positional isomers could not be distinguished from the spectra alone. The hydrolyzed fatty acids were sometimes observed a t a low ir >ensity. The molecular ion is present as a protonated form at m / z 761. Injection of phosphatidylethanolamine resulted in spectra similar to those observed for PC. Diglyceride ions are at m / z 578, and glyceride ions containing 16:O and 18:l fatty acids at m / z 313 and 339, respectively, were observed for the 16:0,18:1-PE (Figure Ib). Instead of the peaks observed at m / z 142 and 184 for phosphocholine, phosphoethanolamine produced peaks at m / z 124 and 141. These peaks were produced by immediate dehydration from protonated phosphoethanolamine and by the loss of water from the ammonium adduct of phosphoethanolamine, respectively. These ions allow unambiguous identification of the phosphate ester head group even when the molecular ions are of low intensity. This information is particularly useful for analysis of a complex mixture. Usually, m / z 142 is monitored for detecting PC species and mlz 124 for P E species, since it is a more intense peak than the 141 peak. The more intense peak a t m / z 124 is due to the instability of protonated phosphoethanolamine.
c
I
200
I00
500
400
300
600
800
700
50
900
MIZ
FI
b
C
0 CHj(CH211,i'C-O-CH2 CHj[CH2$CH:CH
'CH2),-
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R
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on
REL lhl
I
YH. 719
339
141 A
1 I00
200
m
400
500
600
,
I
700
800
wo
1030
M/Z
Flgure 1. Positive ion spectra obtained from direct injection of 2 yg of 16:0,18:l-phosphatidylcholine (a) and 2 yg of 16:0,18:l-phosphatidylethanolamine (b) using 953 methanol:O.1 M ammonium acetate as a carrier solvent. A, B, C7and D in the spectra represent fragment ions generated from cleavage at positions A, B, C, and D, respectively.
In the 16:0,18:1-PE spectrum presented, sodium attachment to the intact molecule was also observed. The intensity of the sodium adducts usually depends on the availability of contaminant sodium ions in the sample, buffer, or ion source. The general pattern of phospholipid fragmentation and the proposed structures of these fragments are presented in Scheme I. Essentially no other significant background peaks were observed. Interpretation of these simple spectra and assignment of phospholipid molecular species are possible in less than 1min. The same type of information could be obtained for a simple mixture after direct injection as well. But for reliable analysis of complex mixtures, the separation of each compound by HPLC before detection is desirable, and this is the subject of the next section. On-Line HPLC/Thermospray MS Analysis. Despite the many HPLC methods previously reported for phospholipid analysis, few methods can resolve individual molecular species of phospholipid in a relatively short time (Le., 30 min). A new reverse-phase chromatographic system that offers rapid analysis and is also compatible with on-line mass spectrometric detection was developed. The separation obtained for a standard PC mixture by the use of this chromatographic
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
Scheme I. Proposed Structure of the Fragment Ions Observed in the Positive Ion Thermospray Spectra F I *. ..,..,,. . C
M /Z
623 624
pj R i C i O - q H 2 ,,,,A %-$'O-CH .;' p
599 600
0 ; CH < O - P - O - C H g C H i R 3
2i
F2
11
575 576
OH
I
I
1680,1882
86.1
625 626 601 602
"
0, ,0-CH2 m/z
,P, 1 .NH; HO O-CH2 0
loo 0
577
142
578 603
22 7
604
627 628
II I
605 606
..-
180,18~1
llB
138 9
TIC h
2 ) PHOSPHATIDYLETHANOLAMINE A
?
H O - P - O - C H ~ ~ N 'HNH ~;-
bn 0 HO- #;o -C H ~ tis C NH~
%H2 B,C,D, FI and F2
0
( R3= -NH2)
H20
I
1
4
8
12
16
20
I
I
24
28
32
TIME( MINI
m h 141
Flgure 3. HPLC Separation of 50 pg of a natural phosphatidylcholine
m/z 124
2. The reconstructed ion chromatograms of digiyceride ions were selected from data acquired by full mass scanning from 120 to 820
mixture from egg yolk obtained under the same condition as in Figure
THE SAME AS FOR PHOSPHATDYLCHOLINE
STANDARD PC MIXTURE, 5 ug REL.INT.
TIC
I
I
I
I
I
I
I
1
0
4
8
I2
16
20
24
20
1
32
TIME(MIN)
Flgure 2. HPLC separation of a standard mixture containing 5 pg of
each synthetic phosphatidylcholine by reverse-phase HPLC using 3-pm Ultrasphere-ODS column. A carrier solvent containing 7 1 5 7 methanol/hexane/O. 1 M ammonium acetate was used at a flow rate of 1 mllmin. The total ion chromatogram was obtalned by full mass scanning from 120 to 820 amu. system is illustrated in Figure 2. Fifty microliters of sample containing 5 pug of each molecular species was injected through a 3-pm Ultrasphere-ODS column (4.6 mm X 7 cm), and the molecular species thus separated were carried directly into the thermospray ion source. This ion chromatogram was obtained by full mass scanning from 120 to 820 amu. The mobile phase was a 71:57 methanol/hexane/O.l M ammonium acetate mixture. Ninety-five percent methanol could also elute phosphatidylcholines, but retention times were longer and poor peak shapes were obtained. By addition of hexane, considerable improvement in analysis time and peak shape
amu. The relative intensity is shown based on the peak height. was achieved. As shown in the ion chromatogram, seven molecular species were separated in 30 min with a flow rate of 1 mL/min. The structures of the individual molecular species were assigned by analyzing the spectrum of each chromatographic peak. It was apparent that they were separated according to their chain length and degree of unsaturation. Coupling HPLC separation and mass spectrometric detection is particularly valuable for the analysis of complex mixtures. An example is shown for a natural phosphatidylcholine preparation from egg yolk (Figure 3). The chromatogram was obtained under the same conditions as in Figure 2. Since the diglyceride ion peaks are predominant in the thermospray spectra of PC, the reconstructed ion chromatograms of only these ions are presented in the upper nine panels along with the total ion current displayed at bottom. Although some species were not completely resolved, selective ion recording of the diglyceride ions allowed deconvolution of the chromatographic peaks. We were able to identify 10 PC molecular species in this 50-pg sample using spectral analysis. In addition, the intensities of these diglyceride ions were close to the reported ratios for PC molecular species in egg yolk (9). This indicated that quantitation based on diglyceride ion intensity or area should be useful for molecular species quantitation even when the chromatographic separation is incomplete. It should also be noted that the precise molecular species composition of different egg yolk phospholipid preparations may vary. The elution pattern also closely matched that previously reported by reverse-phase chromatography obtained under differing LC conditions (IO). Figure 4 illustrates the results for the analysis of a phosphatidylethanolamine standard mixture containing di-18:1, di-182, and di-18:3 species. Fast chromatographic separation of PE species was achieved by using the same ODS column as for PC analysis with a 100:6:5 methanol/hexane/O.l M ammonium acetate mixture as the mobile phase and a flow rate of 1mL/min. Under these conditions, three molecular
12
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
a
a STANDARD PE MIXTURE,
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603 604
0
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DI-18.2-PE(MW 7 3 9 5 )
100
200
300
400
100
600
700
000
900
IC
M/Z
Flgure 5. (a) Total ion chromatogram (mass range 120-800 amu) obtained for 50 k g of phosphatidylethanolamine from egg yolk by reverse-phase chromatography. Conditions were the same as in Figure 4a. (b) Positive ion spectrum obtained from the chromatographic peak assigned as 18:0,18:2-phosphatidylethanolamine in Figure 5a. 100
zoo
3w
400
500
600
mo
800
so0 1000
M/Z
Figure 4. (a) Reverse-phase chromatography of a standard mixture containing di-1&3-, dL182-, and di-18: I-phosphatidylethanolamine using a 3-krn Ultrasphere-ODS column and 100:6:5 methanol/hexane/O. 1 M ammonium acetate. The flow rate was 1 mL/min. The total ion chromatogram (120-820 amu) is presented along with the reconstructed ion chromatograms of the respective diglyceride ions. (b) Positive ion spectrum obtained from the di-l8:2-phosphatldylethanolamine peak in the chromatogram shown in Figure 4a. Water adducts of the glyceride Ion B are observed In the spectrum. F1 and F2 in the spectrum represent the fatty acids released by hydrolysls at positions F1 and F2, respectively.
species were eluted in 10 min, as shown in Figure 4a. The total ion chromatogram shows that a comparable response is obtained from equal amounts of the three species. For phospholipids containing fatty acids with more than one double bond such as di-18:2 or di-18:3, addition of a number of water molecules to the diacylglyceride ions was observed, and this may account for the difference in intensities of these ions. As an example, the spectrum obtained for di-18:2-PE is shown in Figure 4b. In addition to the diacylglyceride ion, fragments with one and two water molecules added are apparent ( m / z 618 and 636, respectively). Formation of these diglyceride-water adducts for the more unsaturated species probably accounts for the decreased intensity of their diglyceride peaks relative to that of the monoene (Figure 4a). Thus, the response of the diacylglyceride ion and the water
adducts should be summed for quantitative studies. For this particular sample the hydrolyzed fatty acid also appeared at mlz 281. This chromatographic system was applied to the analysis of a natural phosphatidylethanolamine preparation from egg yolk (Figure 5). As before, the total ion chromatogram was obtained by full mass scanning from 120 to 820 amu (Figure 5a). With a 50-kg sample injection, at least nine molecular species were separated in 15 min. Again, the distribution of the P E species was estimated from the intensity of the respective diglyceride ions, and the result was in good agreement with previously published data (6). The molecular species separated were identified by analysis of their thermospray spectra. For instance, it was apparent that the peaks at mlz 337 and 341 (Figure 5b) were produced by glyceride ions containing 18:2 and 1 8 0 fatty acids, respectively, and peaks at m / z 124 and 141 confirmed the presence of the phosphoethanolamine head group. The diglyceride ion peak at m / z 604 and the protonated molecule at m / z 745 provided additional support for our assignment of this peak as 18:0,18:2-PE. On-line operation of the HPLC separation with thermospray mass spectral detection reduced the phospholipid analysis time considerably. Even for a complex mixture, unambiguous definition of fatty acyl composition and head group identity of each molecular species can be accomplished
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
13
0 5 p g STANDARD PC MIXTURE, S I M
M/Z 27 s
IS0,ISI
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.
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142.0
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0
200
1
400
I
600
I
800
I
1000
I
1200
360
280
160
TIME (SEC)
Figure 6. Results shown for selected ion monitoring of the molecular ion and major fragments of 14:0,16:O-phosphatidylcholine. Each peak was observed for 100 ms/scan. The specified quantlties were injected by using a 50-pL sample loop without a column. The carrier solvent was 100:65 methanoi/hexane/O.l M ammonium acetate, and the flow rate was 1 mL/min. within 30 min. At the same time, direct quantitation of each molecular species is possible since their thermospray mass spectrometric responses were found to be comparable and reproducible. Detection Limit. Figure 6 shows selected ion monitoring of molecular and fragment ions for 140,160-PC and indicates the present detection limit of this technique. This result was obtained by direct injection (without an HPLC column) using 100:6:5 methanol/hexane/O.l M ammonium acetate as the carrier solvent. Each peak resulted from a separate sample injection using a 50-pL sample loop. Sensitivity could be improved for direct injection by increasing the aqueous solvent proportion in the carrier solvent. Nevertheless, a very low content of water in the mobile phase was necessary for eluting phospholipids from the reverse-phase column as previously demonstrated. The high organic solvent mixture described above was employed here to test detection limits under experimental conditions constrained by those applicable to the analysis of biological sample mixtures. Fifty nanograms of sample was near the lower limit for the simultaneous detection of all fragments including the molecular ion. The deviation in peak areas between replicate analyses was less than 10% even for the 50-ng injection. A similar experiment using the selected ion monitoring technique with an on-line HPLC column is shown in Figure 7 . A mixture containing 0.5 pg of each PC species was separated under the same conditions as for Figure 4a and diglyceride ions were monitored. Inspection of the intensity of early eluting peaks indicates that an acceptable result can be obtained with 5-fold less sample. Postcolumn addition of ammonium acetate buffer may improve the sensitivity of this technique. Care must be taken, however, to prevent clogging the vaporizer tip since phospholipids are not soluble in highly aqueous solutions.
CONCLUSION The use of thermospray mass spectrometry offers significant advantages over conventional techniques for analyzing biological samples, since the direct analysis of intact molecules is possible without derivatization. At the same time this technique also serves as an on-line LC detector while maintaining chromatographic integrity. We describe here a
(17) Kino, M.; Matsumura, T.; Gramo, M.; Saito, K. Biomed. Mass Specfrom. 1982, 9 ,363-369. (18) Dickens, B. F.; Ramesha. C. S.: ThomDson. G. A.. Jr. Anal. Biochem. 1982, 127, 37-48. (19) Crawford, C. G.; Planner, R. D. J. Lipid Res. 1983, 2 4 , 456-460. (20) Crawford, C. G.; Plattner, R. D. J. Lipid Res. 1984, 2 5 , 518-522.
14
Anal. Chem. 1966, 58, 14-19
(21) Lehmann, W. D.; Kessler, M. Chem. Phys. LPMs 1983, 32, 123-135. (22) Wood, G. W.; Lau, P.-Y. Horned. Mass Spectrom. 1974, 1 , 154-155. (23) Sugatani, J.; Klno, M.; Saito, K.; Matsuo, T.; Matsuda, H.;Katakuse, I. Blomed. Mass Spectrom. 1982, 293-301. (24) Aberth, W.; Straubs, K. M.; Burlingame, A. L. Anal. Chem. 1982, 5 4 , 2029-2034. (25) Ohashl, Y. Biomed. Mass Spectrom. 1984, 383-385.
(26) Jungalwala, F. B.; Evans, J. E.; McCluer, R. H. J. LipidRes. 1984, 25, 738-749. (27) Blakley, C. R.; Vestal, M. L. Anal. Ch8m. 1983, 55, 750-752.
RECEIVED for review July 8, 1985. Accepted Seotember 9, 1985.
Short Open Tubular Columns in Gas Chromatography/Mass Spectrometry Michael L. Trehy,' Richard A. Yost,* and John G . Dorsey*
Department of Chemistry, University of Florida, Gainesville. Florida 32611
Short open tubular gas chromatographycolumns can be used with mass spectrometry (MS)for rapld and sensltlve analysls of samples and for the determlnatlon of thermally lablle compounds. The subatmospherlc outlet pressures present wlth mass spectrometric detection result In a slgnlflcant Increase In the optlmum carrler gas veloctty, permmlng extremely rapld analysis. The selectivity avallable wlth MS and particularly with MS/MS methods of analysls Is shown to be sufflclent to determine analytes In complex mlxtures despite the short column length. The feaslblllty of rapld determination of thermally lablle compounds Is demonstrated for the pestlclde aldlcarb and, when on-column Injection Is used, for Its toxlc resldues, aldlcarb sulfoxide and aldlcarb sulfone. Because the analyte concentratlon reachlng the mass spectrometer is greater with shorter columns than wlth longer columns, lower detectlon llmlts are posslble. Bonded-phase columns can be operated at lower temperatures wlthout loss In column efflclency and hence offer slgnlflcant advantages over packed columns for thermally lablle compounds.
The speed of analysis by gas chromatography (GC) is determined by the time necessary for the separation of the analyte from all other mixture components and by the retention time of the last eluting compound. When a highly selective detector such as a mass spectrometer is employed, however, most mixture components will not interfere, and the analysis time can be greatly reduced. As a result, combined chromatographic/spectrometric analysis schemes (the socalled hyphenated methods (1,2))offer considerable promise for the rapid determination of an analyte in complex mixtures. The application of short GC columns with selective detection by mass spectrometry (MS) (3)or by mass spectrometry/mass spectrometry (MS/MS) ( 4 ) makes extremely rapid analysis possible. Furthermore, the short columns provide minimal peak broadening, higher sample concentrations at the detector, and therefore enhanced sensitivity (4). Finally, the use of short open tubular bonded-phase columns permits analysis of compounds too volatile to be introduced by direct probe insertion ( 4 ) , as well as compounds too involatile or thermally labile to be amenable to GC with conventional-length columns (3). Bonded-phase columns can be operated a t lower temperatures without loss in column efficiency and hence are Present address: Monsanto Co., 800 N. Lindbergh Blvd., St.
Louis, MO 63167.
well-suited for the determination of thermally labile compounds. Here the improvements in speed and sensitivity, as well as the analysis of involatile and labile compounds, by mass spectrometry with short open tubular bonded-phase GC columns are discussed.
THEORY The optimum performance of gas chromatography with respect to minimum analysis time is obtained when the outlet is connected to a vacuum (5). The limiting factor in the time required for analysis with thick-film columns is mass transfer in the stationary phase. Improvements in the preparation of open tubular columns have led to thin-film bonded-phase columns with film thicknesses estimated to be approximately 0.1 Km. These thin-film columns significantly increase the rate of mass transfer in the stationary phase, and as a result, the limiting factor with thin-film columns is the rate of mass transfer in the gas phase (6). Sources of Band Broadening in Gas Chromatography. The dependence of the plate height, H on the average gas velocity, u, in open tubular columns is given by the Golay equation (7):
H = B/v
+ Cu
(1)
where B is the coefficient determined by the rate of longitudinal diffusion, and C is the coefficient determined by the rate of mass transfer (8). The increase in plate height that occurs a t carrier gas velocities beyond the optimum velocity often prevents the use of high flow rates, which would decrease the analysis time. Longitudinal Diffusion. The magnitude of the coefficient B in open tubular columns is determined by the diffusion coefficient, DG, in the gas phase.
B =2 0 ~
(2)
DG is in turn dependent on gas pressure (9) DG = 67/5P
(3)
where 7 is the viscosity of the gas and p is the density, as the density of the carrier gas is proportional to both its molecular weight and the pressure applied. Thus, a reduction in the average column pressure or the selection of a carrier gas with a lower molecular weight results in an increase in the gas diffusion coefficient,with a consequent increase in the B term. This increase shifts the optimum velocity to a higher value, which shortens analysis time. The advantage of using light carrier gases such as helium or hydrogen has long been known.
0003-2700/86/0358-00 14$01.50/0 0 1985 American Chemlcal Society
