Anal. Chem. 1988, 60, 1529-1533 ?E
23
I
h
1
'u
c
0:
I
0 2
o n
0 6
0 8
1 0
Flgure 8. Segregation of phosphorus. Process parameters: 30-nm thermai oxide, implantation, 50-keV P, 5 X 10'' om-'. Annealing: 1000 O C , 10 min, O2 -t3% HCi (60-nm SIOz); 1000 O C , 10 min, N;, 400-nm CVD-SO,, 900 O C , 260 min, N., Analytical conditions: 5.5-keV O,', I , = 2 pA, R = 500 X 500 pm2, d A = 60 pm, po, = 5X mbar, mass resoiutlon M/AM = 4300. The depth scale was corrected according to the different erosion rates in Si02and Si. The actual segregation coefficient ms,,s,, 175.
-
ACKNOWLEDGMENT I wish to thank K. Piplits for assistance in SIMS measurements and U. Traxlmayr, E. Guerrero, M. Grasserbauer, and H. Potzl for helpful discussions. The preparation of the samples by the Siemens Research Laboratories in Munich is acknowledged. Registry No. P, 7723-14-0; SiOz, 7631-86-9; Si, 7440-21-3.
LITERATURE CITED (1) Tan, T. Y.; Gijsele, U. Appl. Php. Lett. 1982. 40(7), 616. (2) Hu, S. M. Proceedlngs of the 3rd International Symposium on VLSI
1529
Science and Technology; W. M. Bullis, Ed.; Electrochemical Society, May 1985. (3) Magee, C. W.; Botnlck, E. M. In MRS Symposia Proceedings; Katz, W., Williams, P., Eds.; 1985; Vol. 48, pp 229-240. (4) Stingeder, G.; Grasserbauer, M.;Guerrero, E.:Potzl, H.; Tielert, R. Fresenius' 2.Anal. Chem. 1983, 314, 304. (5) Grasserbauer, M.; Stlngeder, G. TrAC, Trends Anal. Chem. (Pers. Ed.) 1984, 3(5), 133. (6) Grasserbauer, M.; Stingeder, 0.; Potzl, H.; Guerrero, E. Fresenius' 2. Anal. Chem. 1986, 323, 421. (7) Hu, S. M.; Fahey, P.; Dutton, R. W. J . Appl. fhys. 1983, 54(12), 6912. (8) Chu, P. K.; Grube, S. L. Anal. Chem. 1085, 57(6), 1071. (9) Stingerler. G.; Grasserbauer, M.; Traxlmayr, U.; Guerrero, E.; Potzl, H. Mikrochim. Acta, Suppl. 1985, 1 1 , 171. (IO) Huneke, J. C.; Armstrong, T. J.; Wasserburg, G. J. Geochim. Cosmochim. Acta 1883, 47, 1635. (11) McKeegan, K. D.;Walker, R. M.; Zinner, E. Geochim. Cosmochim. Acta lg85, 49, 1971. (12) Stingeder, G.; Piplits, K.; Gara, S.; Grasserbauer, M.; Budil, M.; Potzl, H. SIMS V I , Proceedings of 6th International Conference; Paris, 1987. in press. (13) Lepareur, M. Rev. Tech. Thomson-CSF 1981, 12(1), 225. (14) Wittmaack, K. Appl. fhys. Lett. 1976, 9 , 552. (15) Reuter, W.; Yu, M. L.; Frisch, M. A,; Small, M. B. J . Appl. fhys. 1980, 51(2), 850. (16) Deline, V. R.; Johnson, N. M.; Christel, L. A. Mater. Res. SOC.Symp. froc. 1084, 25, 649. (17) Vandervorst, W.; Shepherd, F. R. Appl. Surf. Sci. 1985, 21, 230. (18) Vandeworst, W.; Shepherd, F. R.; Newman, J.; Phillips, E. F.; Remmerle, J. J . Vac. Sci. Technoi., A 1985, 3(3), 1359. (19) Reuter, W. Nucl. Instrum. Methods fhys. Res., Sect. B 1986, B15, 173. (20) Stingeder, G. Fresenius' 2.Anal. Chem. 1887, 327, 225. (21) Schwarz, S. A.; Barton, R. W.; Ho, C. P.; Helms, C. R. J . Electrochem. SOC. 1881, 128, 1101. (22) Sakamoto, K.; Nlshi, K.; Ichi Kawa, F.; Ushio, S. J . Appl. fhys. 1887, 6 1 , 1553.
RECEIVED for review August 31, 1987. Accepted March 25, 1988. This work has been supported by the Fonds zur Forderung der wissenschaftlichen Forschung (Project No. S 43/10).
Packed Column Supercritical Fluid Chromatography Using Deactivated Stationary Phases M. Ashraf-Khorassani and L. T. Taylor* Virginia Polytechnic Institute and State University, Department of Chemistry, Blacksburg, Virginia 24061 -0212
R. A. Henry Keystone Scientific, Inc., State College, Pennsylvania 16801
A new crosslinked cyanopropyl bonded phase silica (Deltabond) has been studied as a statlonary phase for packed column supercrltlcai fluld chromatography of basic nltrogencontalnlng compounds. The bonded phase Impedes access to uncapped silanol sltes, thereby glvlng rise to better peak shapes and more rapid elutlon without the necessity of a polar modlfler In the mobile phase. Experlments both at elevated temperature and In the presence of a methanol modlfler revealed that there Is no shod- or long-term deleterlous effect on the column as opposed to the conventional cyanopropyl phase.
The acceptance of supercritical fluid chromatography (SFC) by the practicing analytical chemist will be determined in part 0003-2700/88/0360-1529$01.50/0
by the nature and number of applications that can be uniquely addressed by this technique as opposed to other chromatographic methods. While the virtues and problems of capillary and packed column technologies have been espoused for years, most experts now agree that there is need for both. The deveIopment of improved stationary phases for packed columns wherein basic organic analytes are selectively and reversibly partitioned is greatly desired. Currently, commercially available stationary phases suffer from insufficient deactivation of the support surface, which for basic analytes can lead to peak tailing and incomplete elution due to irreversible adsorption. The elution behavior of basic compounds ranging in pK, from -3.0 to +11.0 has recently been inveatigated via SFC with silica, octadecylsilica, and (propy1amino)silica stationary phases and supercritical COz as the mobile phase (I).The 0 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
chomatographic data suggested that silanol-analyte interaction dominated the mechanism of separation, regardless of the stationary phase. Only materials with pK,'s < 1 were eluted from silica, while compounds with pK,'s < 6 were eluted with octadecylsilica. The (propy1amino)silica column exhibited elution of compounds with pK,'s as high as 7.43. These findings were not too surprising, since it has been known for some time that residual silanols are a particular problem in the separation of basic compounds via liquid chromatography and that better surface coverage is provided by the smaller reagent (e.g. octadecyl versus propylamino). Griebrokk and Blilie (2)briefly compared results obtained on a traditional reversed-phase octadecyl column and a CIS column that had been specificallytreated by the manufacturer to remove silanol groups. Polyaromatic hydrocarbons and their nitrated analogues showed much more symmetric peak shapes with the "endcapped" column in the absence of modifier. Several modifiers were examined (e.g. methanol, 1hexanol, tetrahydrofuran, and methyl tert-butyl ether) with the two columns, with the result that the noncapped column demonstrated a greater modifier effect. Kohler and Kirkland (31, however, have noted that only isolated or unbonded acidic SiOH surface groups are largely responsible for the irreversible adsorption of basic molecules in high-performance liquid chromatography (HPLC). They discovered that fully hydroxylated silicas exhibited a larger number of associated silanols and markedly lower adsorptivity for basic compounds. Octyl- and octadecylsilica-bonded phases were demonstrated to show a similar effect. In a related study ( 4 ) a dual retention mechanism that postulates analyte retention as a result of both solvophobic (hydrophobic) and silanophilic interactions was proposed. Surface silanol groups were shown in this investigation to be masked either by increasing the water concentration of the eluent or by the addition of a suitable amine to the eluent so that regular retention behavior was observed in the separation of both crown ethers and peptides. While a more complete silanization reaction may eliminate a significant fraction of isolated silanols, steric considerations would never allow all silanols to be reacted. Schomburg et al. (5) have tried to increase surface coverage by employing a more reactive silanization reagent and by improving access of the reagent to the inside of the silica pores. Immobilization of various kinds of monomers and oligomers in the stationary liquid by cross-linking or thermal peroxide decomposition has also been performed by using methods that have proven successful with gas chromatography (GC) columns. This kind of technology has been applied to SFC only to a limited degree. This manuscript expands on this approach by noting the improved column performance afforded by cross-linking the bonded phase as opposed to non-cross-linking. These new materials, which are referred to as Deltabond columns, are believed to shield any remaining active sites from involvement in the separation mechanism. Compounds of varying polarity have been separated via a cyanopropyl silica-bonded phase employing supercritical COP.
EXPERIMENTAL SECTION A Suprex 200A supercriticalfluid chromatograph that has both capability for density and pressure programming was utilized with a 1 mm i.d. packed column. Restrictors were drawn from short 50 pm i.d. fused silica capillary tubing. The restrictor was connected to the packed column via a zero dead volume adapter (Anspec Co., Ann Arbor, MI). The tip of the restrictor was placed approximately 0.5-2.0cm below the hydrogen/air flow of a flame ionization detector, and the temperature was set at 385 "C to allow expansion of the effluent jet at the tip of the restrictor. A Valco injection valve with 0.1-pL rotor volume was employed for sample introduction to the small-bore column. Supercritical fluid COP (Scott Specialty Gases, Plumsteadville, PA) was pressurized with
1500-psi helium to fill the 250-mL syringe pump. Model compounds employed in this study were obtained from Aldrich Chemical Co. (Madison, WI) and Sigma Chemical Co. (St. Louis, MO). Samples were dissolved in HPLC grade methylene chloride (Fisher Scientific Co., Richmond, VA) prior to injection onto the column. The concentration of samples per component ranged from 2-4 Mg/pL. The columns used in this study were cross-linked cyanopropyl-polysiloxane (Deltabond, Keystone Scientific Co., State College, PA) and regular cyanopropyl, with dimensions of 250 mm X 1.0 mm i.d. and 5-pm particle size diameter.
RESULTS AND DISCUSSION The object of this research was to study the activity and stability of a silica-based cross-linked cyanopropyl bonded stationary phase (Deltabond) with supercritical COz as the mobile phase. A surface coating technique similar to one previously developed for the purpose of capillary GC has been employed (6). T o achieve a less activated surface, the appropriate polysiloxane chains were subsequently linked together by action of a cross-linking agent, thereby effectively masking most of the silanol groups. It should be noted here that capillary SFC columns are predicted to be still more inert when prepared by this procedure, due to substantial differences in the surface areas of the two columns. The cyanopropyl (Deltabond) packed column was tested first to compare its efficiency and activity against a regular cyanopropyl column. Second, the stability of this stationary phase was studied with respect to heat and added methanol. In order to study the activity of the column, a mixture of organic compounds with a wide range of polarity was chosen. The separation of the mixture (e.g. 4 kg/pL each of CI5 hydrocarbon, phenyl acetate, acetophenone, 2,6-dimethylaniline and phenol) on both a regular cyanopropyl and a cross-linked cyanopropyl column are shown in Figure 1. Peak shapes were much improved for the cross-linked phase, especially for 2,6-dimethylaniline, the most basic component. All polar solutes exhibited some tailing on the regular cyanopropyl column, which is probably due to the presence of accessible nonassociated silanol groups or water adsorbed on the silica surface. This type of chromatographic behavior was not observed with the Deltabond column, suggesting that silanol groups are much less accessible and that the partitioning is with the bonded phase. With the regular cyanopropyl column a mixed retention mechanism may be operating, although the retention order is maintained. The difference in t obetween the two separations can be attributed to the fact that the silica base, pore size, and pore size distribution are somewhat different for the two columns. While peak shapes were improved, it was of interest to determine if more polar or basic analytes could be chromatographed with the Deltabond cyanopropyl column. The separation of caffeine has previously been reported, employing a capillary column and 100% COZ at 100 "C (7,8). This same separation on a conventional packed column without modifier a t 160 "C was accomplished with poor efficiency and peak tailing. The separation, however, could be improved with a packed column by going to 5.5% methanol-modified supercritical C02 a t 75 "C (9). Figure 2 shows the separation of caffeine on the cyanopropyl Deltabond with 100% COz at moderate pressure. Excellent efficiency and minimum peak tailing were observed in less than 10 min at both 60 and 100 "C. Previous studies in our laboratory had indicated that tertiary aliphatic amines could not be eluted with supercritical COz from a packed column, regardless of the density and temperature of the mobile phase. Primary and secondary amines naturally would react with COz; therefore, they were not attempted. Employing the cross-linked stationary phase, however, we have been able to elute both triethylamine and
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
A
A
-
B
1
b
loo
i
I
h
h
2
3
4
I
5
I
0
1
1
TIME,
TIME,min
3
I
4
min
Figure 3. Separation of triethylamine (A) and N,Ndimethylbenzylamine (B) on cyanopropyl Deltabond column with 100% CO, and flame Ionization detection at 160 OC.
Table I. Reproducibility of Cyanopropyl Deltabond Column Retention Time and Peak Area
peak
t
L
b
TIME. min. 1 6 0 ' ' ' PRESSURE, alm.
'
1
b
6
6 li '
'
th
1M *
Flgure 1. Separatlon of polarity test mixture on (A) conventional cyanopropyl and (B) cyanopropyl Deltabond column with 100% CO, and flame ionization detection at 60 OC. Key: 1, n-pentadecane; 2, phenyl acetate; 3, acetophenone; 4, 2,6dlmethylaniline; 5, phenol.
60 'C
i
1w 'C
JL 7
TIME man
tRa (% RSD)
K'
CIShydrocarbon phenyl acetate acetophenone 2,6-dimethylaniline phenol
Before Heating 7.29 (0.3) 2.96 7.52 (0.3) 3.09 7.94 (0.3) 3.32 8.55 (0.4) 3.65 10.34 (0.4) 4.62
CI6 hydrocarbon phenyl acetate acetophenone 2,8dimethylaniline phenol
After Heating 7.21 (0.2) 2.92 7.45 (0.2) 3.05 7.85 (0.1) 3.27 8.45 (0.1) 3.59 10.21 (0.3) 4.52
symmetry factor 1.3 2.1
2.4 3.6 4.5 1
1.5 1.8
2.5 3.7
% area
5.3 3.6 2.6 2.9 3.6 4.9 5.9 2.7 4.2 1.2
Average retention time (seven iniections) in minutes. phase. The significant peak tailing accompanying the elution of each amine suggests that the inaccessability of silanol is not complete in this stationary phase. Elution of these two tertiary amines from a regular cyanopropyl column was only possible at modifier levels greater than 10% methanol. The next pa%of our study was concerned with determining the temperature stability and, at the same time, reproducibility of cyanopropyl Deltabond. Seven replicate injections of our polarity test mixture at 60 "C were made, followed by calculation of relative standard deviations for both retention time and peak area. The column was then heated to 150 "C for 12 h with a flow of C02. The column was then brought back to the original operating temperature, and replicate injections were repeated. The results, which are found in Table I, demonstrate very good reproducibility in retention time and peak area both before and after heating for each component. These data suggest that the cross-linked column can be reliably used at relatively high temperature. Parenthetically, the precision in retention time and raw peak areas we have obtained may represent a "worst-case" situation, since we used helium-pressurized COP. Porter et al. (10)have recently reported that precision is much improved when C 0 2without helium head pressure is utilized. However, this hypothesis has been refuted by Schwartz (II), who when working with either capillary or packed columns showed an excellent percentage of relative standard deviation for retention time and raw peak area while employing the helium pump filling method. In point of fact, our data with cyanopropyl Deltabond are in closer agreement with those of
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
I
'I
/I w c
I50
Id0
2 60
PRESSURE, rtm
Flgure 4. Effect of temperature on cyanopropyl Deltabond column: sequential separation of four-component mixture at 60 "C (A), 160 OC (B),and 60 "C (C) with 100% COPand flame ionization detection. Key: 1, diphenylamlne; 2, indole: 3, 7,8-benzoquinoline;4, carbazole. An
identical separation was obtained at these temperatures after a
methanol pretreatment. Schwartz than those of Porter et al., who only used a capillary column. Further corroboration for the stability of the cyanopropyl Deltabond column is seen in the separation of diphenylamine, indole, 7&benzoquinoline, and carbazole, first at 60 "C, then a t 160 "C, and again a t 60 "C. Identical pressure programs were employed in each case, which accounts for the different separation features at the two temperatures (Figure 4). It is noteworthy that the separation improved upon going to the higher temperature and that both separations a t 60 "C were essentially identical. What is even more significant is the results of similar experiments with a regular cyanopropyl column. With the same pressure programming, all four components could be separated at 60 "C albeit with considerable peak tailing, especially for the most basic component, 7,8benzoquinoline (Figure 5). Furthermore, while all peak shapes appear similar for the two runs at 60 "C (Le. before and after heating), the retention time of the 7,8-benzoquinoline has decreased by approximately 0.5 min after heating, in contrast to the other components. The cross-linked cyanopropyl "chemistry" seems to be preferable to conventional endcapping "chemistry" for deac-
150
1so
I ~ L
260
PRcssune. rtm. Flgure 5. Effect of temperature on conventional cyanopropyl column. See Figure 4 for details of separation. tivating silanol groups, on the basis of the following experiment. The regular cyanopropyl column was endcapped with trimethylchlorosilane by the manufacturer. The four-component mixture was separated in the same manner as previously described (Le. 60 and 160 "C; identical pressure programming; same amount injected). The initial 60 and 160 "C runs yielded results that were similar in selectivity to the cross-linked column results. With the temperature decreased to 6O0C,the column performance was similar to the 60 OC runs with the re-glar column. It therefore appears that the elevated temperature caused the trimethylsilyl phase to be removed. The stability of cyanopropyl Deltabond to methanol was next established. Previously in our and other workers' laboratories it has been reported that a methanol modifier leaves a permanent or memory effect on conventional packed column stationary phases, which is most noticeable during the separation of basic analytes. Only after extensive equilibration with COz (24-48 h) could the column be returned to its original state after methanol exposure. Equilibration times of 30-45 min have been noted in ref 2, but these analytes were polyaromatic hydrocarbons and their nitrated analogues not basic materials. An illustration of this phenomenon is provided in Figure 6. A regular cyanopropyl column was washed with approximately 30 mL of methanol followed by equilibration with supercritical COz for 2 h. With the same pressure program and the same four-component mixture previously de-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
n
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those previously shown in Figure 4. In the case of the regular column, the limited liquid methanol pretreatment apparently activates the stationary phase by converting siloxane units to silanols, which after COz equilibration are more numerous than before methanol pretreatment. Heating the column no doubt removes a fraction of the silanol units, thereby creating a less active column that facilitates the elution of 7,8-benzoquinoline. With cyanopropyl Deltabond these reactive siloxane groups are not accessed by the liquid methanol; consequently, the activity of the column is unchanged. These results should not be construed to mean that the cyanopropyl Deltabond column is without a modifier effect. An optimized separation of the same four components was accomplished by employing 100% COz followed by identical/pressure programming but with a mobile phase of either CO2/CH3OH (99/1) or COz/CH30H (98/2). The three nonbasic nitrogen-containing compounds eluted with a small change in retention time regardless of the mobile phase. The presence of methanol caused 7,8-benzoquinoline to elute at shorter retention times. In fact, with 2% methanol, 7,8-benzoquinoline eluted before indole. From these results with a cross-linked cyanopropyl column, the cross-linking of the surface of bonded phases, in general, could be a step toward the further advancement of packed columns for both SFC and HPLC. Also, other types of cross-linked packed columns (CI8 C8, C1, NHz, phenyl, and poly(ethy1ene glycol)) are currently under preparation and evaluation.
n
ACKNOWLEDGMENT Without the loan of a supercritical fluid chromatograph by Suprex Corp. much of this work could not have been performed. Special thanks go to Keystone Scientific, Inc., for providing all columns employed in this study.
LITERATURE CITED
~~
150
GO
NO
pacssuac. atm. Floure 6. Effect of methanol pretreatment on conventional cyanopropyl column at 60 and 160 "C. See Figure 4 for details of separation.
scribed, 7,8-benzoquinoline could, surprisingly, not be eluted. The same mixture when injected at 160 "C revealed now four peaks, wherein 7,8-benzoquinoline exhibited much peak tailing. Upon return of the column to 60 "C the efficiency for the most basic compound had changed, as evidenced by the appearance of a peak ascribable to 7,8-benzoquinoline7 which originally at 60 "C did not elute. An identical experiment with the cross-linked cyanopropyl phase (i.e. 30 mL of methanol, 2 h, COz equilibration) showed no varying effect due to methanol pretreatment. The results were identical with
(1) Ashraf-Khorassanl, M.; Taylor, L. T., submitted for publlcation as a Huethig Publishing, Ltd., Monograph. (2) Blllle, A. L.; Grelbrokk, T. Anal. Chem. 1985, 57, 2239. (3) Kahler, J.; Kirkiand, J. J. J. Chromafogr. 1987, 385, 125. (4) Bij, K. E.; Horvath, C.; Meiander, W. R.; Nahum, A. J. Chromatogr. 1987, 203, 65. (5) Schomburg, G.; Deege, A,; Kohler, J.; Bien-Vogeisang, V. J. Chromatogr. 1983, 282, 27. (8) Biomberg, L. C.; Markides, K. E.; HRC CC, J . High Resoluf. Chrometogr. Chromafogr. Commun. 1985, 8 , 632. (7) Yarbro, S. K.; Gere, D. R. Chromafography, 1987 (April), 49. (8) Later, D. W.; Richter, B. E.; Knowles, D. E.; Anderson, M. R. J. Chrometogr. Sci. 1986, 2 4 , 249. (9) Gere, D. R. Hewiett-Packard Application Note, an 800-6, 1983. (IO) Porter, N. L.; Richter, B. E.; Bornhop, D.J.; Later, D. W.; Beyerlein, F. H. HRC C C , J. High Resoluf. Chromafogr. Chromatogr., Commun. 1987, IO, 477. (11) Schwartz, H. E.; Barthel, P. J.; Moring, S. E. HRC CC, J. High Resoluf. Chromafogr. Chromafogr ., Commun . 1987, 10 668. ~
RECEIVED for review December 14, 1987. Accepted April 6, 1988. The financial assistance by Department of Energy Grant DE-FG22-81PC40799 is greatly appreciated.
