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achieving a well-deactivated surface. The ability to attach a variety of pendant groups to the hydrosiloxane backbone to achieve a wide range of selective phases adds to the future importance of this method for preparing packed columns for chromatography.
LITERATURE CITED (1) Sander, L. C.; Wise, S. A. Crit. Rev. Anal. Chem. 1987, 78, 299-417. (2) RoumeliOtiS. P.; Unger, K. K. J. Chromatogr. 1976, 725, 115-127. (3) Sander, L. C.; Wise, S. A. Anal. Chem. 1984, 56, 504-510. (4) Majors, R. E.: Hopper, M. J. J. Chromatogr. Sci. 1974, 72,767-778. (5) Verzeie, M.; Mussche, P. J J. Chromatogr. 1983, 254, 117-122.
(6) Figge, H.; Deege, A,: Kohler, J.; Schomburg. G. J. J. Chromatcgr. 1986. 351, 393-408. (7) Woolley, C. L.; Markides, K. E.: Lee, M. L. J . Chromatogr. 1986, 367,
9-22. (8) Woolley, C. L.; Markides, K. E.;Lee, M. L. J. Chromatogr. 1988, 367, 23-34. (9) Iler, R. K . The Chemistry of Silica: Wlley: New York, 1979; Chapter 6. (10) Walters, M. J. J . Assoc. Anal. Chem. 1987, 70, 465-469.
RECEIVED for review December 5, 1989. Accepted March 26, 1990. This work was supported by the Gas Research Institute, Contract No. 5088-260-1640,and the State of Utah Centers of Excellence Program, Contract No. 87-2279.
Multidimensional Packed Capillary Coupled to Open Tubular Column Supercritical Fluid Chromatography Using a Valve-Switching Interface Z. Juvancz,' K. M. Payne, K. E. Markides, and M. L. Lee* Department of Chemistry, Brigham Young University, Provo, Utah 84602
An on-line twwtiiensional supercritical fluid Chromatographic system (SFCISFC) was constructed by utilizing a rotary valve interface to provide independent flow control of the two dimensions. A cold trap was employed to refocus solutes from $Ingle or multiple fractional cuts, after being transferred to the second dimension. Improved performance, including time savlngs, was achieved with a packed capillary to open tubular column arrangement and two independently controlled pumps, compared to earlier reported slnglegump open tubular column SFCISFC and packed Capillary column SFCISFC systems. The packed capillary in the first dimension provided a rapid chemical class Separation, while the open tubular column in the second dimension provided high resolution of closely related isomers.
INTRODUCTION Samples for chemical analysis often contain so many components, that only a multidimensional chromatographic system can approach their complete separation ( I , 2). Furthermore, high molecular weight samples have exponentially increasing numbers of isomers, requiring even greater resolution than their low molecular weight counterparts. Numerous reports have been published describing multidimensional chromatography using GC (3),LC (4,SFC (5),and their combinations (6-8). Since SFC is a high-resolution technique and is suitable for separation of high molecular weight samples, it is a natural choice for combining in a multidimensional configuration. An operational advantage of multidimensional SFC/SFC is that the transferred sample from the first dimension can be easily refocused. At atmospheric pressure in the focusing region, the mobile phase instantly loses its solvating power. On leave from t h e C e n t r a l Research I n s t i t u t e f o r Chemistry of the Hungarian Academy of Sciences, H 1025 Budapest, Pusztaszeri ut 59-67, Hungary. * Corresponding author.
This solubility loss is enhanced by the cooling effect of the expanding carrier, resulting in effective precipitation of the compounds of interest in a narrow band. The problems associated with elimination of the carrier from the first dimension is, therefore, much easier to solve in SFC than in LC. Packed columns can provide faster analysis times and higher sample capacities than open tubular columns. A recent paper describing a multidimensional packed capillary SFC/SFC system (9) reported short analysis times but insufficient efficiency in the second dimension to separate many closely related isomers. The properties of packed capillary columns, however, make them an excellent choice for use in the first dimension. Open tubular columns with their high resolving power are, on the other hand, the column type of choice for the second dimension. Multidimensional open tubular column SFC /SFC has been shown ( I O ) to be a promising, but time consuming, technique for resolving complex mixtures. I t was already proposed (9) that a suitable compromise between speed and resolution could be reached by using a packed capillary column for a faster group type separation in the first dimension and an open tubular column in the second dimension to provide the final high-resolution separation of individual compounds. Due to the significant differences in the optimum volumetric flow rates of packed capillary and open tubular columns, earlier attempts to combine a packed capillary to an open tubular column for multidimensional SFC/SFC have failed because the flow rates could not be independently controlled. In this paper, a two-dimensional packed capillary to open tubular column SFC/SFC system is reported that uses a valve-switching interface in line with a cryogenic trap for refocusing of analyte fraction cuts in the second dimension. This interface allowed independent flow-rate control of the two columns, providing a simple means for selective transfer of one or more solute fractions to the second dimension. The system was evaluated by using a certified coal tar that had been used previously for evaluating open tubular column SFC/SFC and packed capillary SFC/SFC (9, IO).
0003-2700/90/0362-1384$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15. 1990
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Flgum 1. Sdrematic diagram of a multidimensional packed capillary to open tubular column SFClSFC system. Abbreviations: Col, = primary packed capillary column; Col, = secondary open tubular column: V, and V, = rotary valves: T = cold tap: S = capillary solute concentrator; R, and R, = fri reshlctm: R, = linear restrictor; Z = zero dead volume union: B = bun connector. The text contains the detailed description of Um system.
EXPERIMENTAL S E C T I O N Instrumentation and Materials. A schematic diagram of the packed capillary to open tubular column SFC/SFC system is shown in Figure 1. It consisted of two Series 501 syringe pumps, two Series 600 controllers, and two SFC/GC ovens with flame ionization detectors (Lee Scientific, Salt Lake City, UT). SFC grade liquid CO, (Scott Specialty Gases, Plumsteadville, PA) was delivered to the injector through a 1.5-m X 1Wpm i.d. fused silica capillary (all fused silica tubing used in this study was purchased from Polymicro Technologies, Phoenix, AZ). Liquid injections were made using a pneumatically actuated injector with a 2WnL internal injection loop (Valco. Houston, TX). An untreated IO-cm X 100-pm i.d. fused silica capillary was used as a flow connection line between the injector and the packed capillary column (Col,). Zero dead volume unions (Valco) were used to connect the packed capillary column in the system. A 60-cm X 250rm i.d. fused silica capillary column (Col,) was packed with 7-rm particle diameter, 300-A pore, aminosilanebonded silica (NH,-silica; Nucleosil, Macherey-Nagel, DCiren, FRG) with a stainless steel microscreen (Mectmn, City of Industry, CA) to support the packed bed (9). A 20-cm x 1Wpm i.d. fused silica capillary connected Col, to a IO-port, 2-position valve, V, (Valco). This valve was used to direct the flow of the effluent to either the fust FID (FID,) through restrictor R, (15cm x 50pm of i.d. frit restrictor, Lee Scientific), or to the transfer line (b) the second dimension. The rotors of valves V, and V, were ordered to meet specificationsof 150 OC and 6Mx) psi so that they could be operated under SFC conditions inside the oven. The transfer line & (1.2-m X 15pm i.d. fused silica capillary) served &salinear restrictor between V, and the cold trap. The end of & was placed inside a deactivated fused silica capillary in the middle of the cold trap (ScientificGlass Engineering, Austin, TX). The basic concept and working parameters of the cold trap have been described in detail elsewhere (11.12). Liquid CO, was used for the cooling of the trap to 5-15 OC during trapping. A 15-em x 200-pm i.d. deactivated fused silica capillary (Lee Scientific) or solute concentrator, S, was placed between the cold trap and the column of the second dimension (Col,). The column in the second dimension (Col,) was a 10.5-m x %rm i.d. fused silica open tubular column coated with a liquid crystalline polysiloxane stationary phase (SB-SmecticdO; Lee Scientific). Butt connectors (Scientific Glass Engineering) were used to connect Col, in the system. A frit restrictor (&) was used between Col, and FID,. A 10-port valve (V,) was used to direct the m i e r from Pump,, either through VI and the transfer line, &, or directly to Col, through the cold trap. Small segments of 200-pm4.d. Teflon tubing (Chrompack. Middelhurg, The Netherlands) were used to eliminate potential dead volumes in the injector, V,, and the unions, 2. A coal tar (Standard Reference Material, SRM 1597; National Institute of Standards and Technology, Gaitherburg, MD) and
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a polychlorinated biphenyl sample (Arochlor 1242; Supelco, Bellefonte, PA) were used to demonstrate the operation of the system. Operating Procedure. The samples were injected without splitting onto the packed capillary column. Isothermal pressure programs were used to elute the sample components from the fust dimension. The beginning and ending of the cutting periods were determined from a previous run of the sample under identical conditions. For the studies reported in this paper, the instrumentation was operated as described in the following paragraphs. A t the time of injection, VI directed the effluent flow from Col, to FID,, and V, directed the flow of Pump, a t 414 atm directly to the cold trap. The vent and the cooling liquid CO, flows were off. Two minutes before the cut, V, was turned, changing the direction of the COP carrier from Pump, to V, through R, and the cold trap. The eoolmg liquid CO, flow was switched on, and the vent was opened. A t the time of the cut, V, was turned to transfer the target analytes from Col, to the cold trap. V, was then turned back, allowing the eluent from Pump, to purge through R, for an additional 3 min a t 414 atm into the cold trap to ensure complete transfer. The pressure of Pump, was then brought down to 70 atm. Approximately 3 min was required to decrease the pressure from 414 to IO atm. As smn as Pump, reached 70 atm, the vent valve and cooling liquid CO, were turned off and V, was changed to direct the carrier from Pump, directly to the cold trap. At the same time, the second oven was switched on. When the temperature of the second oven reached the preset value (after approximately 3 m i d the pressure program was started and the chromatogram from the second dimension was developed.
RESULTS AND DISCUSSION T h e use of a capillary to carry the mobile phase effluent from Pump, to the injector prevented hackflushing of the sample and helped to avoid peak splitting during injection. T h e sample capacity of Col,, which was =2 pglcomponent (13),allowed enough sample to be injected for the analysis of minor components in the second dimension from only a single fractional cut. This is an important advantage over a 50-pm-i.d. open tubular column (0.25-rm film thickness), which has a sample capacity of only 75 ng/component. In addition, the system also allowed multiple injections in the fmt dimension with repetitive collection of the same fractional cut for enrichment of trace analytes. The flow rate used in Col, met the flow requirements of FID, (9). However, the time saved in the analysis was the main reason why the packed capillary was chosen for the first dimension. T h e time required for the analysis in the first dimension of a coal tar (SRM1597) took 2.3 h when an open tubular column SFC (IO) was used, compared to 55 min when a packed capillary column was used (Figure 2). Even though the packed capillary column could not provide the same resolution as an open tubular column, it did provide a selective group-type separation from which fractions could be cut and transferred to the second dimension. The aminosilane stationary phase used in the first column provided a good separation of the coal tar extract according to the number of aromatic rings. This packed capillary column produced adequate separation for sharp fractional cuts. The retention pressures of the peaks varied by only 1-2 atm. The eluting peaks had acceptable peak shapes up to six aromatic rings. Components having six and seven rings eluted with some peak broadening, caused by low solubility of these high molecular weight compounds in C02and the loss of efficiency at higher mobile-phase densities. The rotory valve interface allowed independent flow control of each dimension, which provided a "time-efficient" and flexible method for joining the two dimensions. The packed capillary column open tubular column configuration is used in this study, but this interface could also he used in other column combinations, with or without solute focussing between the two dimensions. After a fractional cut was com-
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Figwe 2. Packed capillary column chromatogram (Col,)of a standard coal tar (SRM 1597). Conditions: CO,; 75 O C pressure program from 200 to 414 atm at 5 atm min-’.
pleted, both dimensions could be run in parallel. While the shape-selective separation of isomers was being done in the second dimension, Coll could be cleared of late eluting components. A total of 57 min was needed to separate and identify the chrysene/triphenylene isomeric pair and to clear Col, for the following analysis. This should be compared to 3 h that was required to do the same analysis with the open tubular column SFCjSFC system using an offset cross interface and one carrier source (10). It was not possible to make such a complete separation with packed capillary SFC/SFC (9). Another potential advantage of the valve switching interface is that by avoiding solvent peak transfer or transfer of other high-concentration components, it is possible to broaden the range of usable detectors in the second dimension. By introduction of only the peaks of interest into the second dimension, it becomes favorable to use, for example, a mass spectrometer as a second detector. Major matrix component peaks that might contaminate the detector can be avoided. Applying two independent pumps also provides the option of using different mobile phases for each dimension. These options could increase the selectivity differences between the two dimensions to a great extent. Minimizing the dead volume was an important consideration in this design. Teflon tubing spacers were used in the injector, valve connections, and zero dead volume unions in the first oven (13). Care had to be taken to avoid extending the capillaries too far into the valves where they could scratch the rotor. If the capillaries, on the other hand, were not inserted far enough into the Teflon tubing, the compressed sleeves could plug the flow. A 10-port rotary valve was used for the fractional cuts because it had shorter loops (less dead volume) than a valve with fewer ports. No memory effects were observed from valve VI in either FID, or FID,. The transfer line linear restrictor, R2,was adjusted to produce a similar restriction as R,. In this way, negligible peak shifting was observed for later eluting peaks on Col,. This can be seen by comparing the chromatograms in Figures 2 and 3A. The flow and pressure conditions were unchanged in Col, during the cutting periods. These periods were made 20% longer than the elution bands of selected fractions in order to reduce the potential of cutting errors.
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Figure 3. Two-dimensional SFC chromatograms of a standard coal tar (SRM 1597). Two fractions were collected from the first dimension and then analyzed simultaneously in the second dimension. Cut ”a” was collected between 20.2 and 21.2 min, and cut “b” between 38.7 and 40.2 min. Conditions: (A) same as in Figure 2; (6) CO,; 110 O C ; pressure program from 70 to 120 atm at 20 atm min-’, and then 120 to 414 atm at 8 atm min-’, after a 2-min isobaric period. Peak identifications: (1) triphenylene, (2) chrysene, (3) benzo[ghi]perylene, (4) anthracene. After cutting, the fraction was transferred to the cryogenic trap. High pressure was needed during this transfer, because the mobile phase had a negative density gradient along the linear restrictor, RP. The loss of solvating power over the length of the transfer line was compensated for by an extra long transfer period and by using high pressure from Pump2. A study was performed to investigate the length of time required for total transfer of fractions. Peak areas were
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Flgure 4. Two-dimensionalSFC chromatograms of a standard coal tar (SRM 1597). The sample was run twice through Coil; identical fractions were collected in the cold trap and then analyzed on Col,. Condltions: same as in Figure 3. Peak identification: (1) triphenylene, (2) 6-methylchrysene, (3) chrysene, (4) 1-methylchrysene, (5) 2methyichrysene.
measured as a function of transfer period. When the peak areas reached a constant value, total transfer of the fraction was presumed. I t was thus experimentally determined that the time of 1.5 min was needed to transfer sample components of up to six aromatic rings, while 3 min was needed for coronene. As mentioned earlier, there was no memory effect observed from the transfer line. I t can be concluded that the system allowed complete transfer of all Components studied and that the chromatography was not limited by the interface between valve V1 and the cold trap. The Hon-columnncryogenic trap, used in this study, provided new possibilities for the analysis of multicomponent mixtures by SFC. The efficiency losses from the first dimension, due to dead volumes and multiple connections, could be completely compensated for by the efficient trapping of selected fractions and subsequent release of the solutes in a narrow band onto the second column. The configuration of the trap with open vent made it possible to transfer and refocus the effluents from the first dimension without disturbing the flow in Col,. Typical volumetric flow rates for packed (250-pm id.) capillary columns are on the order of 10-30 mL min-'. This is in contrast to 50-pm4.d. open tubular columns, which have volumetric flow rates on the order of 0.1-1.0 mL min-'. While the difference in these flow rates prevented the use of the offset-cross interface (9, IO), the independent flow control offered by the rotory valve/cold trap interface allowed both columns to be operated within their respective working ranges. Both the cooling of the trap and the depressurization of carrier through the vent helped to produce a very sharp solute band inside the trap. The temperature of the trap was not completely optimized for light molecules. Trapping of semivolatile compounds like anthracene could, however, be made
50 I
70
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Figure 5. Two-dimensional SFC chromatograms of a polychlorinated biphenyl (Arochlor 1242) sample. Conditions: (A) CO,; 90 O C ; presswe program from 130 to 414 atm at 3 atm min-'; = heart-cut peak between 29 and 30 min. (B) CO,; 110 OC; pressure program from 70 to 120 atrn at 20 atm min-', and then 120 to 414 atm at 5 atm min-', after an initial 2-min isobaric period.
with only a slight loss in recovery. Compounds from pyrene to coronene gave 100% recovery, and the separations were highly efficient. The 100% trapping efficiency made it possible to collect several fractions, for example, from the same run on Col, and subsequently analyze them in one run on Col2. Also, the same fraction could be collected from several runs on Col, and then be analyzed on Co12,enhancing trace level components. Sequential analysis of more than one fraction is demonstrated in Figure 3 by the collection of two fractions from an analysis on Col, and then their analysis in one run on Colz.
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No recovery or efficiency losses were observed, and the elution conditions of the second fraction were not disturbed by the first cut. A total of 85 min was needed for the combined separation of both the chrysene/triphenylene and anthanthrene/ benzo[ghi]perylene isomeric pairs in this system. No peak broadening was observed from longer collection times. The different cuts were selected such that no overlap of the fractions would occur in the second dimension. The elution region of cut “a” was clearly resolved from the region of cut “b“ in Figure 3B. Trapping of the same fraction from repetitive separations on Col, followed by analysis of this compiled fraction in one run on Col, is a useful technique for trace analysis. The quantitative trapping efficiency of the cold trap allowed the trapped components to be effectively stored over the period of multiple runs. The trace carcinogenic 1-, 2-, and 6methylchrysenes were detected in an SRM 1597 coal tar extract in this manner (Figure 4). In this case, two runs were needed to collect enough sample (1-10 ng) for detection by FID? Col, did not separate chrysene from the methylchrysene isomers in the first dimension; however, the large chrysene peak did not disturb the elution of these other compounds. No sample loss or peak distortion was observed by performing multiple collections. If several cuts were collected in the cold trap, the high-pressure transfer of sample components to the trap from R2 was needed only after the last collection step. This routine conserved analysis time and minimized the background from trapped impurities in the mobile phase. Use of the capillary solute concentrator was originally developed to maximize inertness for almost “on-column” trapping of adsorptive solutes (11,12). This short length of deactivated fused silica tubing was inserted between the incoming restrictor and Col,, allowing the incoming restrictor to be inserted =3 cm into the solute concentrator, maximizing trapping efficiency and inertness. This configuration also minimized peak-splitting and peak-broadening. The capillary solute concentrator provided phase ratio focusing at the connection to Col,, which compensated for noninstantaneous release of materials from the cold trap. However, the initial parameters in the second dimension had to be chosen carefully, even when the capillary solute concentrator was used. The oven temperature was kept under 60 “C during the trapping period in order to keep the cold trap cool enough for precipitation of solutes. The oven was then raised to 110 “C during the analytical run, because the best resolution of PAH on the liquid crystalline column was achieved a t temperatures above 90 “C (14). The mobile phase was maintained in the gaseous state (70 atm) during heating of the second oven from 60 to 110 OC in order to limit the mobile phase solvating power during this period. This low starting pressure provided the additional advantage of minimizing the pressure wave that was created on Col:, at the closing of the vent.
The smectic liquid crystalline polysiloxane stationary phase was a good choice for the second dimension. Its shape-selective properties were different from the selectivity of the aminosilane phase of the first dimension. This combination was found to be useful for the analysis of polychlorinated biphenyls (PCB, Figure 5) as well as for PAH isomers. Many PCB isomers have similar volatilities and polarities, and they differ from each other mainly by their shapes. In these cases, a shape selective phase is the best candidate to separate them. Figure 5A shows the separation of arochlor 1242 on Col, according to polarity, and Figure 5B shows the shape selective separation of the components in a single peak from the first dimension. The SB-Smectic-50 stationary phase is much more selective at 110 “C in SFC than at the higher elution temperature for these compounds necessary in GC (14). In summary, the mild conditions that can be used for multidimensional SFC and the enhanced selectivity at these low temperatures make it an attractive alternative to multidimensional GC for thermally labile and closely related solutes. In addition, the interface is easy to use for high molecular weight solutes.
ACKNOWLEDGMENT We thank Stephen A. Wise (U.S. National Institute of Standards and Technology) for helpful discussions with respect to SRM 1597 and, especially, Ilona Davies (Dionex) for discussions relating to the construction and practice of multidimensional SFC.
LITERATURE CITED Giddings, J. C. Anal. Chem. 1984, 56, 1258A-1270A. Davies, I.L.; Raynor, M. W.; Kithinji, J. P.; Bartle, K. D.; Williams, P. T.; Andrews, G. E. Anal. Chem. 1988, 60, 683A-702A. Schomburg, G. LCIGC 1987, 5, 304-317. Gluckman, J. C. J . Chromatogr. Libr. 1985, 30, 57-72. Campbell, R. M.; Djordjevic, N. M.; Markides, K. E.; Lee, M. L. Anal. Chem. 1988, 6 0 , 356-362. Cortes, H. J.; Pfeiffer, C. D.; Jewett, G. L.; Richter, B. E. J . Microcol. Sep. 1989, I . 28-34. &ob, K. I n Tenth International Conference on CaplUary Chromatcgraphy; Sandra, P., Ed.; Huethig: Heidelberg, 1989; pp 465-471. Bartle, K. D.; Davies, I.L.; Raynor, M. W.; Clifford, A. A,; Kithinji, J. P. J . Microcol. Sep. 1989, 7 , 63-70. Payne. K. M.; Davies, I. L.; Bartle, K. D.; Markides, K. E.; Lee, M. L. J . Chromatogr. 1989, 477, 161-168. Davies, I. L.; Xu, B.; Markides, K. E.; Bartle, K. D.; Lee, M. L. J . Microcol. Sep. 1989, l , 71-84. Xie, Q. L.; Markides, K. E.; Lee, M. L. J . Chromatogr. Sci. 1989, 27, 365-370. Koski, I . J.; Jansson. E. A,; Markides, K . E.; Lee, M. L., unpublished results. Payne, K. M.; Tarbet, E. J.; Bradshaw, J. S.;Markides, K. E.; Lee, M. L. Anal. Chem., preceding paper in this issue. Chang, H.-C. K.; Markides, K. E.; Bradshaw, J. S.;Lee, M. L. J . Chromatogr. Sci. 1988, 2 6 , 280-289.
RECE~VED for review December 5, 1989. Accepted March 26, 1990. This work was supported by the Gas Research Institute (Contract No. 5088-260-1640).
