2680
Anal. Chem. 1986,58,2680-2683
capacity factor for a series of alkylbenzenes vs. volume fraction of acetonitrile. It is clear that the larger solutes deviate from the line defined by the results from 50 to 90% modifier to a greater extent as the test solute increases in size.
ACKNOWLEDGMENT The authors also thank C. Reichardt for sharing his data on the ET^ values in mixed aqueous solvents. Registry No. H20,7732-18-5;benzene, 71-43-2;fluorobenzene, 462-06-6; chlorobenzene, 108-90-7; bromobenzene, 108-86-1; toluene, 108-88-3; iodobenzene, 591-50-4; rn-dichlorobenzene, 541-73-1;o-dichlorobenzene,95-50-1;p-dichlorobenzene, 106-46-7; ethylbenzene, 100-41-4; m-xylene, 108-38-3; o-xylene, 95-47-6; mesitylene, 108-67-8;isopropylbenzene,98-82-8;o-diethylbenzene, 135-01-3; durene, 95-93-2; tert-butylbenzene, 98-06-6; benzyl alcohol, 100-51-6;2-phenylethanol, 60-12-8; dimethylbenzyl alcohol, 29718-36-3; 3-phenylpropanol, 122-97-4; nitrobenzene, 98-95-3;benzaldehyde, 100-52-7;phenol, 108-95-2;p-chlorophenol, 106-48-9;p-nitrophenol, 100-02-7;acetonitrile, 75-05-8. LITERATURE CITED Sadek, P. C.; Carr, P. W.; Doherty, R. M.; Kamlet, M. J.; Tan R. W.; and Abraham, M. H. Anal. Chem. 1985, 57, 2971-2978. Taft, R. W.; Abraham M. H.; Doherty R. M.; Kamlet, M. J. Nature (London) 1985, 313,384. Taft, R. W.; Abraham, M. H.; Famini, G. R.; Doherty, R. M.; Abboud, J. L.; Kamlet, M. J. J. Pharm. Sci. 1985, 74,807. Melander, W. R.; Campbell, D. E.; Horvath, Cs. J . Chromatogr. 1978, 158. 215. Melander, W R.; Chen, B.-K.; Horvath. Cs. J . Chromatogr. 1979, 185, 99.
(6)Melander. W. R.; Chen, B.-K.; Horvath. Cs. J. Chromatogr. 1985, 378, 1. (7) Melander, W. R.; Horvath, Cs. Chromatographia 1984, 353, 18. (8) Snyder, L. R. J. Chromatogr. 1979, 179,167. (9) Gant, J. R.; Dolan, J. W.; Snyder, L. R. J. Chromatogr. 1979, 785, 153. (10) Dec, S. F.; Gill, S. J. J. Chem. f d u c . 1985, 62,879. (11) Yonker, C. R.; Zwier, T. A.; Burke, M. F. J. Chromatogr. 1982, 2 4 4 , 257. (12) Yonker. C. R.; Zwier, T. A,; Burke, M. F. J . Chromatogr. 1982, 244, 269. (13) Gilpin, R . K.; Squires, J. A. J. Chromatogr. Sci. 1981, 79,195. (14) Laurance, C.; Nicolet, P. J. Chem. Soc., Perkin Trans. 2 ,in press. (15) Xindu, G.; Regnier, F. E. J. Chromatogr. 1985, 332, 147. (16) Weisberg, S. Applied Linear Regression; Wiley: New York, 1980. (17) Nahum, A.; Horvath, Cs. J. Chromatogr. 1981. 203,33. (18) Jinno, K.; Kawasaki, K. J. Chromatogr. 1984, 376, 1. (19) Sadek, P. C.; Carr, P. W.; Bowers, L. D. J. Li9. Chromatogr. 1985, 8 , 2359. (20) Krygowski, T. M.; Reichardt, C.; Wronka, P. K.; Wyszomirska, C.; Zielkowska, U. J. Chem. Res. 1983, 116. (21) Krygowski, T. M.; Wronka, P. K.; Zielkowska, U.; Reichardt, C. Tetrahedron 1985, 47,4519. (22) Antle, P. E.; Goldberg, A. P.; Snyder, L. R. J. Chromatogr. 1985, 321, 1.
RECEIVED for review March 18, 1986. Accepted June 9, 1986. Work at the University of Minnesota was supported by a grant from the National Science Foundation. The work by R. W. Taft was supported in part by a grant from the Public Health Service as was the work at Yale University. The work by M. J. Kamlet and R. M. Doherty was done under Naval Surface Weapons Center Task IR-060.
Liquid Chromatography-Gas Chromatography Interfacing Using Microbore High-Performance Liquid Chromatography with a Bundled Capillary Stream Splitter Thomas V. Raglione and Richard A. Hartwick* Department of Chemistry, Busch Campus, Rutgers University, Piscataway, New Jersey 08854
An on-line LC-GC system Is presented. HPLC peak volume reductlon was accompllshed through the use of mlcrobore (1.0 mm) columns. Further volume reduction was accomplished by use of a novel bumlled multlcaplllarystream spWtter between the LC detector and the GC. Appllcatlons of the system for the separatkn of solvent-refined coal samples are presented. ReproduclblWtles of the dlrect and split systems were 13 % and 9 % relathre standard deviatlon, respectlvely.
True multidimensional separations offer fundamental advantages over single-mode techniques in the separation of complex mixtures (1,2). Many GC analyses of environmental and biomedical samples directly or indirectly depend upon off-line LC for sample cleanup and/or class fractionation. The coupling of HPLC to high-resolution GC is thus an attractive instrumental design that should find applications in many areas of analysis. Interest in developing efficient HPLC-GC interfaces is not new. Majors (3) and Apffel and McNair ( 4 ) were among the first to attempt on-line coupling by employing conventional HPLC systems coupled to capillary GC systems. However, due to the large eluted peak volumes associated with conventional (4.6 mm i.d.) HPLC systems, it was only possible to heart-cut a small fraction of the LC peak to the GC, making 0003-2700/86/0358-2680$0 1.50/0
quantitation difficult. Nevertheless, this early work clearly demonstrated the feasibility and power of the technique, as well as the areas that needed improvement. Grob et al. (5, 6) employed an on-column concentration technique coined “retention gap” as a means of improving quantitation by permitting the transfer of larger peak volumes to the GC. Although it was possible to transfer up to 1mL of eluent, the length of retention gap required (approximately 50 m) resulted in solvent evaporation times of up to 1 h. Munari et al. (7) noted the drawbacks of such long retention gaps and investigated the use of shorter (10 m) gap lengths. The use of these shorter retention gaps places restrictions on the solutes of interest as well as the LC mobile phase. Concurrent solvent evaporation requires that the solutes of interest elute a t least 50 OC above the boiling point temperature of the mobile phase. Reversed-phase separations would have limited applicability due to the high-boiling aqueous mobile phases employed (8). Though the retention gap technique is time-consuming and has limited compatibility with reversed-phase LC, it is a valuable technique especially when applied to trace analysis. More recently, in an attempt to more closely match the volume requirements of liquid and gas chromatography, Cortes et al. (9, IO) have successfully interfaced packed capillary (300 pm i.d.) HPLC with capillary GC using the retention gap method, However, miniaturization of the HPLC to this level Q 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
permits only submicroliters of instrumental dead volume, making the routine implementation of such instrumentation technically demanding. This is especially relevant in LC-GC, where instrument size often dictates longer than desired transfer lines. The retention gap technique for zone reconcentration partially overcomes these problems; however, in an ideal situation, it would be desirable to have a more flexible approach open to LC instrument design. The purpose of the present work was to calculate the basic instrumental requirements of LC-GC instrumentation and to study alternatives to complete instrument miniaturization. Microbore HPLC (1.0 mm id.) columns were chosen as a practical working alternative to packed capillary columns due to their more widespread use and commercial availability. Further LC zone volume reduction was accomplished via a novel isokinetic eluent splitter designed to reduce the LC peak volumes to those compatible with GC injection volumes.
EXPERIMENTAL SECTION Instrumentation. A Varian Model 5500 LC (Varian Assoc., Walnut Creek, CA) equipped with a UV 200 detector and a 0.5-pL flow cell was used for these experiments. Injections were made by triggering a pneumatic Rheodyne valve 7126 (Rheodyne,Cotati, Ca) for the appropriate time interval. LC columns (1.0 X 250 mm) were prepared by slurrying 300 mg of ODS-bonded Whatman silica gel (Whatman, Inc., Clifton, NJ) in 3 mL of 2-propanol and packing at 10000 psi. The mobile phase was methanol/water (80:20). A Varian 3400 capillary GC equipped with a FID was used for these experiments. The GC was operated with a split of 701. A 16-m SE-30 column was used, and the carrier gas was helium. The oven was held at 90 “C for 2 min, then ramped at 5 OC/min to 250 O C . The interface consisted of a modified Varian GC autosampler equipped with a 10-pL flow-through syringe. The transfer volume was kept constant at 10 pL. In the direct mode 16 cm of 0.009-in. Teflon tubing was used as a transfer line from the LC detector to the interface. In the split mode 8 cm of 0.009-in. Teflon tubing was used to connect the splitter and the interface. All solvents were HPLC grade, and the pyrene was spectroscopic grade. The liquid coal tar (SRCII heavy distillate 3317015-23C) was generously supplied by C. Wright of Battelle Pacific Northwest Labs. Bundled Fiber Splitter Design. Splitters were constructed by epoxying 25-pm fused silica capillaries into a short length of 0.040-in.-i.d. ‘/16-in. stainless-steel tubing then randomly selecting the desired split ratio by taking a fraction of these capillaries and epoxying them into a short length of 0.020-in.4.d. 1/16-in.tubing. RESULTS AND DISCUSSION Peak Transfer Requirements. One of the major problems in the design of LC-GC interfaces has been achieving acceptable reproducibility. Conventional (4.6 mm id.) HPLC columns, with peak elution volumes 2 orders of magnitude larger than the required injection volumes for either packed or capillary GC, offer inherent problems for on-line LC-GC systems. Thus, in designing a direct transfer interface the GC injection volume dictates the size of the transferred volume, and thus the requirements of the LC system. These requirements can be calculated with reasonable accuracy from basic theory. The area percent of the HPLC peak transferred to the GC vs. capacity factor (k’)of the peak was calculated for a series of HPLC column diameters (4.6,2.0, 1.0,0.5 mm). Constant column length (25 cm) and plate efficiency (10000) were assumed. A 10-pL transfer volume was used. The results of these calculations are presented in Figure 1. As shown in Figure 1, 4.6-mm-i.d. columns transfer only a few percent of an LC peak to the GC. Small-bore columns of 2.0 mm transfer 30-50% of an HPLC peak at k’ values of less than 1,dropping off sharply to less than 20% for k’ values of 2 or more. Total zone transfer over a significant range of
I
3
5
capacity factor (k’)
2681
7
Flgure 1. Effect of column internal diameter on the percentage of peak area transferred vs. the capacity factor of the transferred LC zone. Calculations assume an LC column length of 25 cm, producing 10 000 plates with a constant transfer volume to the GC of 10 1L.
k’ values can be achieved with column diameters of ca. 0.5 mm i.d., as shown by the top curve of Figure 1. Microbore (1mm i.d.) columns under similar conditions will transfer over 80% of a peak at k’ < 2 and 45% of the peak a t k’ = 5. Column diameters of between 1 and 0.5 mm i.d. could be considered as suitable candidates for LC-GC interfacing designs by subjectively choosing minimum transferred areas of about 75% or more. Even under gradient elution conditions, with peaks eluting a t a velocity of k’ = 2,2-mm4.d. columns will not transfer sufficient peak area to permit good reproducibility of the LC zone, which by definition will consist of many peaks and possibly include an internal standardb). An advantage of using smaller column diameters is the minimization of the error associated with the sampling time. At conventional flow rates, 1.0 mL/min, 10 pL is approximately equal to 0.5 s, so any slight changes in retention due to mobile-phase evaporation, flow rate variation, sample concentration variation, stationary-phase hydrolysis, etc., will drastically affect the reproducibility of such a system. Similarly, with microbore flow rates, 50 pL/min, 10 pL is approximately 12 s, thus minimizing the effect of retention changes. While the area percent of the HPLC peak transferred will affect reproducibility, instrument sensitivity will concurrently be affected by the actual samples mass transferred. These calculations are summarized in Figure 2, which shows the transferred mass (sensitivity) of an arbitrary HPLC peak vs. capacity factor, given the same column efficiencies as in Figure 1 and a constant column loading in terms of micrograms of solute per milligrams of packing. I t is interesting that the mass transferred from the LC to the GC is less strongly dependent upon LC column diameter than the volume transfer, since peaks from the larger diameter columns are being heart-cut onto the GC. At a k’ = 2, the sensitivity would be similar for LC columns of 1.0, 2.0, or 4.6 mm i.d. In contrast, a 0.5-mm-i.d. column of similar efficiency would yield a sensitivity 3.5 times less than this. It is evident from Figures 1 and 2 that a trade-off between peak transfer area and sensitivity must be made in designing the LC-GC system. Further considerations not specifically addressed here include extracolumn dispersion. I t was felt that 1.0-mm-i.d. microbore columns offered a reasonable compromise between system sensitivity and peak transfer volumes. Microbore columns and instruments are commercially available and are far less demanding than packed capillary columns in terms of transfer lines and other dispersion
2682
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
CAPiLLARY G C O F COAL T A R I O * 0 5
0
*OJ\\
I
5
3
I
7
capacity factor ( k ' )
Ftgwe 2. Effect of column diameter on sensitivity. Plot of micrograms transferred from the LC to the GC vs. capacity factor of the zone. Columns are the same as listed in Figure 1 with a constant sample loading (micrograms of sample per milligrams of packing material).
MICRO-IC O F
0
COAL T A R
4
8
12
16
20 24
28min
Flgure 4. Coal tar sample directly injected into the GC equipped with
a FID. The GC was operated in the split mode with a 70:l split. The GC oven was held at 90 OC for 2 min and then ramped at 5 'C/min to 250 OC
MICROBOHL HPLC/CAPII 1 4Ri G C
1
O
0'1
02
03
04
06m1
j
Figure 3. Coal tar sample injected on a 1.O X 250-mm ODS column: mobile phase, 80:20 methanol/H,O; detection at 254 nm.
sources in the system. It is also clear from these figures that column diameters greater than 1.0 mm i.d. are almost certainly too large for any type of direct, Le., nonsplit, interfacing with a GC. By use of the instrumentation described in the Experimental Section, the reproducibility of the direct transfer of the microbore LC zone to the GC was evaluated using pyrene as a test solute, eluting a t h' = 3 on the LC column, with a retention time of 24 min on the capillary GC. Replicate injections produced a relative standard deviation averaging 13% for the entire system. Applications of Direct LC-GC Interfacing. The amount and identification of polynuclear aromatic hydrocarbons (PAH) in coal tar has been the subject of considerable research (I1,12). Such a complex matrix could directly benefit from the selectivity gains of multidimensional LC-GC. A solvent-refined coal sample (SRC I1 3317-015-23C) was injected directly on both the microbore LC and capillary GC in Figure 3 and 4, respectively. The work of Giddings (13)
Figure 5. Direct LC-GC analysis of pyrene in the coal tar sample. The LC and GC were operated under the same conditions listed in Figures 3 and 4. The transferred zone is depicted on the LC chromatogram.
and others regarding the deconvolution of complex chromatograms suggests that many of the peaks in Figure 4 probably include numerous constituents. The peak capacity of the GC column has essentially been saturated, with very few chromatographic regions available for further separation. In contrast many of the compounds in the coal tar sample went undetected by the UV detector on the LC system since they lack chromophores (see Figure 3). Figure 5 shows the same coal tar sample injection on the direct LC-GC system. Although the multidimensional system was able to easily separate pyrene from the other components in the sample, heart-cutting of the LC zones occurred due to the widths of the eluted zones, despite the use of microbore (1.0 mm) columns. Splitter Design and Rationale. The ultimate solution to the problem of dissimilar volume requirements of two interfaced systems, whether they be LC-GC, LC-MS, or LC-LC, would seem to be in the design of a low-dispersion isokinetic splitter (14). Since an ideal splitter would horizontally slice the zone of interest, discrimination due to
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
I
2:l SPLIT RATIO
COAL TAR FBAC ON
m Figure 6, LC-GC analysis of the coal tar sample with eluent splitter: eluent split ratio, 1:2. Operating conditions were the same as in Figures 3 and 4.
heart-cutting effects would be minimized. In real packets of zones, volumes substantially larger than ideal will be encountered, since it may be desired to transfer a significant range of peaks over to the secondary system. In such cases, even packed capillary LC columns can be too large for the primary instrument. A properly designed splitter effectively decouples volume considerations of the primary and secondary systems. Sensitivity, however, will not improve over a micro-LC column when using sample splitters. The amount of solute injected in a splitter experiment will essentially follow the curves shown in Figure 2, where the split fraction might be equal to a normal peak on a 0.5-mm, 1.0-mm, etc., column. Typical splitter designs employ two flow channels, a split channel and a waste channel. To minimize mixing, the linear velocities in the two channels should be identical. Since the adjustment is normally accomplished through some type of needle valve or calibrated orifice, the linear velocities in the two channels will often be different. Also, it can be difficult to maintain constant split ratios under changing experimental conditions, with the restriction devices being prone to plugging, wear, etc. A novel splitter design, employing bundled open tubular capillaries, was developed from a previous bundled capillary HPLC column designed in our laboratory (15). Linear velocities between the capillaries are directly proportional to the length and inversely proportional to the square of the diameter. While the diameter requirements are very stringent for efficient open tubular HPLC, they are easily achieved in practice for an HPLC splitter. The bundle can be readily bifurcated, with the split ratio being determined by the number of fibers used in each leg. In principle, any number of split channels can be fabricated. Such a splitter could simultaneously transfer a single LC or GC peak to several detectors and/or other separation modes, each with different volume requirements. Applications of LC-GC with Splitter. A prototype splitter was constructed by epoxying 36 (25 wm x 15 cm)
2683
capillaries into a short length of 0.040-in.-i.d. 1/16-in.stainless-steel tubing. By randomly selecting 12 of these capillaries and epoxying them into a short length of 0.020-in.4.d. 1/16-in. tubing a split of 1:2 was achieved. Reproducibility of this splitter was determined using pyrene (k’= 3). Using a 1:2 split relative standard deviation averaged 9 % , a 40% increase compared with the direct injection microbore system under identical conditions. Figure 6 shows the separation of the same coal tar sample injected on the LC-GC system, employing a 1:2 split. The 1:2 split allows the GC to effectively sample a 30-pL slice of the zone. Thus, by tailoring the split ratio to the elution volume, a representative slice of the zone may be transferred to the GC, eliminating the discrimination errors associated with heartcutting. In summary, an interface utilizing eluent splitting appears to us to represent the most flexible approach to multidimensional interfacing of column chromatographic techniques, such as LC-GC and LC-LC, since the volume requirements of the primary and secondary systems become independent of one another. The alternatives to this are to heart-cut a primary peak, which in terms of quantitation represents probably the worst possible approach, or to force a match in volume between both systems, which restricts the versatility of design and in some cases rules out the use of an entire current generation of instrumentation (i.e., conventional HPLC). Eluent splitters, if properly designed, would allow the interfacing of a conventional LC column to a GC without the need for a retention gap. Sensitivity, however, will be lost when using any splitter interface. Thus, for trace analysis some type of on-column concentration or zone compression (16)would be preferred. Nevertheless, an interface utilizing a splitter is well-suited for a wide range of routine applications due to its speed and versatility.
ACKNOWLEDGMENT We thank Ron Majors of Varian Associates and Gerald Dupre of Exxon Research for their helpful discussions on various aspects of this work. LITERATURE CITED (1) Giddings, J. C. Anal. Chem. 1984, 56, 1259A. (2) Ragllone, T. V.; Sagllano, N., Jr.; Floyd, T. R.; Hartwick, R. A. LC-GC Mag. 1986. 4 , 328. (3) Majors, R. E. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. 1979; paper 116. (4) Apffel, J. A.; McNalr, H. J . Chromafogr. 1983, 279, 139. (5) Grob, K . , Jr. J . Chromafogr. 1982, 237, 15. (6) Grob, K., Jr.; Frolich, D.; Schilling, 8.; Neukom, H. P.; Nageli, P. J . Chromatogr. 1984, 295, 55. (7) Munari, F.; Triscianl, A.; Mapelli, G.; Trestianu, S.; Grob, K., Jr.; Colin, J. M. HRC CC , J High Resolut. Chromafogr . Chromatogr . Commun. 1985, 8, 801. (8) Grob, K., Jr.; Schilling, B. HRC CC, J . High Resolut. Chromafogr. Chromatogr. Commun. 1985, 8 , 726. (9) Cortes, H. J.; Pfelffer, C. D.; Rlchter, B. E . HRC CC, J . High Resoluf. Chromafogr Chromafogr. Commun. 1985, 8 , 469. (10) Cortes, H. J.; Richter, B. E.; Pfelffer, C. D.; Jensen, D. E. J . Chromatogr 1985, 349, 55-6 1. (1 1) Wright, C. W.; Weimer, W. C.; Springer, D. L. Chromafographla 1984, 18, 603. (12) Hertz, H. S.; Brown, J. M.; Chesler, S. N.; Guenther, F. R.; Hiipert, L. R.; May, W. E.; Parris, R. M.; Wise, S. A. Anal. Chem. 1980, 52, 1650. (13) Davis, J. M.; Giddings, J. C. Anal. Chem. 1985, 57, 2166. (14) Dupre, G. D. Exxon Research Corp., personal communication. (15) Meyer, R. F.; Champlin, P. B.; Hartwick, R. A. J . Chromatogr. Sci. 1983, 21, 433. (18) Hsien, S. H.; Ragiione, T. V.; Tomellini, S. A.; Floyd, T. R.; Sagliano, N. Jr.; Hartwick, R. A. J . Chromafogr. 1988, 367, 293.
.
.
.
RECEIVED for review May 5,1986. Accepted July 7,1986. The support of the donors of the Petroleum Research Fund, administered by the American Chemical Society, a Biomedical Research Support Grant, and the donation of the LC-GC instrumentation by Varian Associates are gratefully acknowledged.
